Implantable biocompatible immunoisolatory vehicle for delivery of selected therapeutic products

ABSTRACT

An immunoisolatory vehicle for the implantation into an individual of cells which produce a needed product or provide a needed metabolic function. The vehicle is comprised of a core region containing isolated cells and materials sufficient to maintain the cells, and a permselective, biocompatible, peripheral region free of the isolated cells, which immunoisolates the core yet provides for the delivery of the secreted product or metabolic function to the individual. The vehicle is particularly well-suited to delivery of insulin from immunoisolated islets of Langerhans, and can also be used advantageously for delivery of high molecular weight products, such as products larger than IgG. A method of making a biocompatible, immunoisolatory implantable vehicle, consisting in a first embodiment of a coextrusion process, and in a second embodiment of a stepwise process. A method for isolating cells within a biocompatible, immunoisolatory implantable vehicle, which protects the isolated cells from attack by the immune system of an individual in whom the vehicle is implanted. A method of providing a needed biological product or metabolic function to an individual, comprising implanting into the individual an immunoisolatory vehicle containing isolated cells which produce the product or provide the metabolic function.

RELATED APPLICATIONS

This application is a continuation of Ser. No. 10/007,344 filed Oct. 25,2001 now abandoned, which is a continuation of Ser. No. 09/563,248 filedMay 2, 2000, now U.S. Pat. No. 6,322,804, which is a divisional of Ser.No. 09/148,671 filed Sep. 4, 1998, now U.S. Pat. No. 6,083,523, which isa divisional of Ser. No. 08/449,837 filed May 24, 1995, now U.S. Pat.No. 5,874,099, which is a divisional of Ser. No. 08/179,151 filed Jan.10, 1994, now U.S. Pat. No. 5,800,828, which is a continuation-in-partof PCT application Ser. No. U.S. 92/03327 filed Apr. 22, 1992, which isa continuation-in-part of Ser. No. 07/692,403 filed Apr. 25, 1991, nowabandoned.

BACKGROUND

Many clinical conditions, deficiencies, and disease states can beremedied or alleviated by supplying to the patient a one or morebiologically active moieties produced by living cells or removing fromthe patient deleterious factors which are metabolized by living cells.In many cases, these moieties can restore or compensate for theimpairment or loss of organ or tissue function. Examples of disease ordeficiency states whose etiologies include loss of secretory organ ortissue function include

-   (a) diabetes, wherein the production of insulin by pancreatic islets    of Langerhans is impaired or lost;-   (b) hypoparathyroidism, wherein the loss of production of    parathyroid hormone causes serum calcium levels to drop, resulting    in severe muscular tetany;-   (c) Parkinsonism, wherein dopamine production is diminished; and (d)    anemia, which is characterized by the loss of production of red    blood cells secondary to a deficiency in erythropoietin. The    impairment or loss of organ or tissue function may result in the    loss of additional metabolic functions. For example, in fulminant    hepatic failure, liver tissue is rendered incapable of removing    toxins, excreting the products of cell metabolism, and secreting    essential products, such as albumin and Factor VIII. Bontempo, F.    A., et al., Blood, 69, pp. 1721-1724 (1987).

In other cases, these biologically active moieties are biologicalresponse modifiers, such as lymphokines or cytokines, which enhance thepatient's immune system or act as anti-inflammatory agents. These can beparticularly useful in individuals with a chronic parasitic orinfectious disease, and may also be useful for the treatment of certaincancers. It may also be desirable to supply trophic factors to apatient, such as nerve growth factor or insulin-like growth factor-oneor -two (IGF1 or IGF2).

In still other cases, the biologically active moiety can be a secretorysubstance, such as a neurotransmitter, neuromodulator, hormone, trophicfactor, or growth factor, or a neuroactive substance for the reductionof pain sensitivity. Such neuroactive substances include catecholamines,enkephalins, and opioid peptides.

In many disease or deficiency states, the affected organ or tissue isone which normally functions in a manner responsive to fluctuations inthe levels of specific metabolites, thereby maintaining homeostasis. Forexample, the parathyroid gland normally modulates production ofparathyroid hormone (PTH) in response to fluctuations in serum calcium.Similarly, β cells in the pancreatic islets of Lang rhans normallymodulate production of insulin in response to fluctuations in serumglucose. Traditional therapeutic approaches to the treatment of suchdiseases cannot compensate for the responsiveness of the normal tissueto these fluctuations. For example, an accepted treatment for diabetesincludes daily injections of insulin. This regimen cannot compensate forthe rapid, transient fluctuations in serum glucose levels produced by,for example, strenuous exercise. Failure to provide such compensationmay lead to complications of the disease state; this is particularlytrue in diabetes. Jarret, R. J. and Keen J., (1976) Lancet(2):1009-1012.

Many other diseases are, likewise, characterized by a deficiency in abiologically active moiety that cannot easily be supplemented byinjections or longer-term, controlled release therapies. Still otherdiseases, while not characterized by substance deficiencies, can betreated with biologically active moieties normally made and secreted bycells. Thus, trophic and growth factors may be used to preventneurodegenerative conditions, such as Huntington's and Alzheimer'sdiseases, and adrenal chromaffin cells which secrete catecholamines andenkephalins, may be used to treat pain.

It is also fairly well established that the activation of noradrenergicor opioid receptors in the spinal cord by direct intrathecal injectionof α-adrenergic or opioid agonists produces antinociception, and thatthe co-administration of subeffective doses of these agents can producepotent analgesia. The presence of enkephalin-secreting neurons and opiatreceptors in high densities in the substantia gelatinosa of the spinalcord and the resultant analgesia observed following local injection ofopiates into the spinal cord have suggested a role for opioid peptidesin modulating the central transmission of nociceptive information. Inaddition, catecholamines also appear to be important in modulating painsensitivity in the spinal cord since injection of noradrenergic agonistsinto the subarachnoidal space of the spinal cord produces analgesia,while the injection of noradrenergic antagonists produces increasedsensitivity to noxious stimuli.

Many drugs have been administered intraspinally in the clinical setting,and numerous methods are available to deliver intraspinal medications.For instance, the most common method of intraspinal drug delivery,particularly anesthetics, is continuous infusion by way of spinalcatheters. However, the use of these catheters, particularly small-borecatheters, has been implicated in such complications as cauda equinasyndrome, a neurological syndrome characterized by loss of sensation ormobility of the lower limbs. In fact, the FDA was prompted to issue asafety alert in May, 1992, alerting Anesthesia Care Providers to theserious hazard associated with continuous spinal anesthesia bysmall-bore catheters and has taken action to remove all small-borecatheters from the market.

Accordingly, many investigators have attempted to reconstitute organ ortissue function by transplanting whole organs, organ tissue, or cellswhich provide secreted products or affect metabolic functions. Moreover,transplantation can provide dramatic benefits but is limited in itsapplication by the relatively small number of organs suitable andavailable for grafting. In general, the patient must be immunosuppressedin order to avert immunological rejection of the transplant, whichresults in loss of transplant function and eventual necrosis of thetransplanted tissue or cells. In many cases, the transplant must remainfunctional for a long period of time, even for the remainder of thepatient's lifetime. It is both undesirable and expensive to maintain apatient in an immunosuppressed state for a substantial period of time.

A desirable alternative to such transplantation procedures is theimplantation of cells or tissues within a physical barrier which willallow diffusion of nutrients, waste materials, and secreted products,but block the cellular and molecular effectors of immunologicalrejection. A variety of devices which protect tissues or cells producinga selected product from the immune system have been explored. Theseinclude extravascular diffusion chambers, intravascular diffusionchambers, intravascular ultrafiltration chambers, and implantation ofmicroencapsulated cells. Scharp, D. W., et al., World J. Surg., 8, pp.221-9 (1984)2. These devices would alleviate the need to maintain thepatient in an immunosuppressed state. However, none of these approacheshave been satisfactory for providing long-term transplant function. Amethod of delivering appropriate quantities of needed substances, suchas enzymes, hormones, or other factors or, providing other neededmetabolic functions, for an extended period of time is still unavailableand would be very advantageous to those in need of long-term treatment.

SUMMARY OF THE INVENTION

This invention relates to a biocompatible, immunoisolatory, implantablevehicle. The instant vehicle is suitable for isolating biologicallyactive cells or substances from the body's protective mechanismsfollowing implantation into an individual. The instant vehicle iscomprised of (a) a core which contains isolated cells, either suspendedin a liquid medium or immobilized within a hydrogel matrix, and (b) asurrounding or peripheral region (“jacket”) of permselective matrix ormembrane which does not contain isolated cells, which is biocompatible,and which is sufficient to protect the isolated cells in the core fromimmunological attack.

The immunoisolatory vehicle is useful (a) to deliver a wide range ofbiologically active moieties, including high molecular weight products,to an individual in need of them, and/or (b) to provide needed metabolicfunctions to an individual, such as the removal of harmful substances.The instant vehicle contains a multiplicity of cells, such thatimplantation of a few or a single vehicle is sufficient to provide aneffective amount of the needed substance or function to an individual. Afurther advantage offered by the instant vehicle is practicality ofretrieval.

In one embodiment of the invention, which is particularly useful withislets of Langerhans, both the core and the surrounding or peripheralregion of the instant vehicle are hydrogels, which can be the samecomposition hydrogel or different composition hydrogels.

This invention also relates to a method of delivering a biologicallyactive moiety or altering a metabolic or immunologic function in anindividual in need of the moiety or altered metabolic function. Animmunoisolatory vehicle of the present invention is implanted into theindividual (referred to as the recipient), using known techniques ormethods and selected for the particular immunoisolatory vehicle and siteof implantation. Once implanted, cells isolated within the biocompatibleimmunoisolatory vehicle produce the desired moieties or perform thedesired function(s). If moieties are released by the isolated cells,they pass through the surrounding or peripheral permselective membraneor hydrogel matrix into the recipient's body. If metabolic functions areprovided by the isolated cells, the substances to be metabolized (e.g.,degraded or inactivated) enter the vehicle from the recipient's body andare removed from the recipient's bloodstream.

Thus, this invention relates to a method of isolating cells within abiocompatible, immunoisolatory implantable vehicle, thereby protectingthe cells within the vehicle from immunological attack after beingimplanted into an individual. Although some low molecular weightmediators of the immune responses (e.g. cytokines) may be permeable tothe membrane, in most cases local or circulating levels of thesesubstances are not high enough to have detrimental effects. The isolatedcells are protected from attack by the recipient's immune system andfrom potentially deleterious inflammatory responses from the tissueswhich surround the implanted vehicle. In the core of the vehicle, theisolated cells are maintained in a suitable local environment. In thismanner, needed substances or metabolic functions can be delivered to therecipient even for extended periods of time, and without the need totreat the recipient with dangerous immunosuppressive drugs.

This invention relates further to a method of making a biocompatibleimmunoisolatory vehicle. In a first embodiment, the vehicle is formed bycoextruding from a nested-bore extrusion nozzle materials which form thecore and surrounding or peripheral regions, under conditions sufficientto gel, harden, or cast the matrix or membrane precursor(s) of thesurrounding or peripheral region (and of the core region). A particularadvantage of this coextrusion embodiment is that the cells in the coreare isolated from the moment of formation of the vehicle, ensuring thatthe core materials do not become contaminated or adulterated duringhandling of the vehicle prior to implantation. A further advantage ofthe coextrusion process is that it ensures that the surrounding orperipheral region is free of cells and other core materials. Thepermeability and biocompatibility characteristics of the surrounding orperipheral region are determined by both the matrix or membraneprecursor materials used, and the conditions under which the matrix ormembrane is formed.

In another embodiment of the present method, the immunoisolatory vehicleis formed stepwise. For example, if the immunoisolatory vehicle beingmade includes a hydrogel core containing the isolated cells, the corecan be formed initially, and the surrounding or peripheral matrix ormembrane can be assembled or applied subsequently. Conversely, thesurrounding or peripheral matrix or membrane can be preformed, and thenfilled with the preformed isolated-cell containing core material or withmaterials which will form the core (i.e., core precursor materials). Thevehicle is sealed in such a manner that the core materials arecompletely enclosed. If a core precursor material is used, the vehicleis then exposed to conditions which result in formation of the core.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphic representation of the differences in thepermeability of alginate matrices formed under different conditions totest solutes of various molecular sizes.

FIG. 2 is a graphic representation of the results of a perfusion test ofthe functionality of immunoisolated versus unprotected islets maintainedin vitro for four weeks. FIG. 2A depicts the results obtained with animmunoisolatory vehicle having a hydrogel core matrix and a peripheraljacket made of a permselective thermoplastic membrane. FIG. 2B depictsthe results obtained with an immunoisolatory vehicle having a hydrogelcore matrix and a peripheral hydrogel jacket.

FIG. 3 is a graphic representation showing the total amount of insulinreleased, and the amounts released during the first and second phaseresponses in the perfusion test also shown in FIG. 2. FIG. 3A depictsthe results obtained with the dual-matrix immunoisolatory vehicle andFIG. 3B depicts the results obtained with the core matrix-permselectivemembrane immunoisolatory vehicle.

FIG. 4 is a graphic representation of the decrease in plasma glucoselevels observed when immunoisolated xenogeneic islets are implanted intostreptozotocin-induced diabetic mice for a period of 60 days. Theimmunoisolatory vehicle used was of the configuration described inExample 5.

FIG. 5 is a graphic representation of insulin release in a perfusionexperiment using an immunoisolatory vehicle containing rat islets, ofthe configuration described in Example 5, recovered after a period ofresidence in vivo and challenged with glucose, with and withouttheophylline stimulation.

FIG. 6 is a graphic representation of the decrease in plasma glucoselevels observed when immunoisolated xenogeneic islets are implanted intostreptozotocin-induced diabetic mice for a period of 100 days. Theimmunoisolatory vehicle used was of the configuration described inExample 4.

FIG. 7 is a graphic representation of the permeability of an alginatematrix to various test solutes. Permeabilities were tested after storagein Hank's solution after 16 hours and 160 hours. The change inpermeability is due to leaching of Ca⁺⁺ from the matrix.

FIG. 8 is a graphic comparison of the response to glucose challenge ofrat islets isolated within dual matrix immunoisolatory vehicles witheither thermoplastic or alginate jackets, following a period ofresidence in vivo in discordant xenogeneic recipients (guinea pigs).

FIG. 9 is a graphic representation of the partial restoration of normalmotor behavior to rodents with experimentally induced Parkinson-likebehavior, following implantation of an immunoisolatory vehiclecontaining adrenal chromaffin cells in a core matrix, with a surroundingjacket of permselective thermoplastic membrane.

FIG. 10 is a graphic representation of the mean body weight changes seenin quinolinic acid lesioned rats. Rats receiving immunoisolatorycapsules containing bovine adrenal chromaffin cells maintained bodyweight significantly better than the other lesioned rats.

FIG. 11 is a graphic representation of the nonfasting plasma glucoseconcentrations of diabetic mice after implantation of type 2 acryliccopolymer hollow fibers containing either 1000 rat islets (A) or 500 ratislets (B) implanted either intraperitoneally (circles) orsubcutaneously (squares).

FIG. 12 is a graphic representation of the effects on blood glucose indiabetic rats implanted with rat islets encapsulated in flat sheetdevices.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to a biocompatible immunoisolatory vehiclesuitable for long-term implantation into individuals. More particularly,the biocompatible immunoisolatory vehicle of the instant inventioncomprises (a) a core which contains a biologically active moiety, eithersuspended in a liquid medium or immobilized within a hydrogel orextracellular matrix, and (b) a surrounding or peripheral region ofpermselective matrix or membrane (jacket) which does not containisolated cells, which is biocompatible, and which is sufficient toprotect isolated cells if present in the core from immunological attack.

The term “individual” refers to a human or an animal subject.

A “biologically active moiety” is a tissue, cell, or other substance,which is capable of exerting a biologically useful effect upon the bodyof an individual in whom a vehicle of the present invention containing abiologically active moiety is implanted. Thus, the term “biologicallyactive moiety” encompasses cells or tissues which secrete or release abiologically active molecule, product or solute; cells or tissues whichprovide a metabolic capability or function, such as the removal ofspecific solutes from the bloodstream; or a biologically active moleculeor substance such as an enzyme, trophic factor, hormone, or biologicalresponse modifier.

When the biologically active moiety within the core of the biocompatibleimmunoisolatory vehicle comprises cells, the core is constructed toprovide a suitable local environment for the continued viability andfunction of the cells isolated therein. The instant vehicle can be usedto immunoisolate a wide variety of cells or tissues, spanning the rangefrom fully-differentiated, anchorage-dependent cells or primary tissues,through incompletely-differentiated fetal or neonatal tissues, toanchorage-independent transformed cells or cell lines.

Many transformed cells or cell lines are most advantageously isolatedwithin a vehicle having a liquid core. For example, PC12 cells (whichsecrete dopamine and are herein shown to be useful for the treatment ofParkinsonism) can be isolated within a vehicle whose core comprises anutrient medium, optionally containing a liquid source of additionalfactors to sustain cell viability and function, such as fetal bovine orequine serum.

Unless otherwise specified, the term “cells” means cells in any form,including but not limited to cells retained in tissue, cell clusters,and individually isolated cells.

Implants of the vehicle and contents thereof retain functionality forgreater than three months in vivo and in many cases for longer than ayear. In addition, the vehicle of the current invention may be preparedof sufficient size to deliver an entire therapeutic dose of a substancefrom a single or just a few (less than 10) implanted and easilyretrievable vehicles.

The core of the immunoisolatory vehicle is constructed to provide asuitable local environment for the particular cells isolated therein. Insome embodiments, the core comprises a liquid medium sufficient tomaintain the cells. Liquid cores are particularly suitable formaintaining transformed cells, such as PC12 cells. In other embodiments,the core comprises a gel matrix which immobilizes and distributes thecells, thereby reducing the formation of dense cellular agglomerations.The gel matrix may be composed of hydrogel or extracellular matrixcomponents.

Suitably, the core may be composed of a matrix formed by a hydrogelwhich stabilizes the position of the cells in cell clumps. The term“hydrogel” herein refers to a three dimensional network of cross-linkedhydrophilic polymers. The network is in the form of a gel substantiallycomposed of water, preferably but not limited to gels being greater than90% water. Cross-linked hydrogels can also be considered solids becausethey do not flow or deform without appreciable applied shear stress.

Compositions which form hydrogels fall into three classes for thepurposes of this application. The first class carries a net negativecharge and is typified by alginate. The second class carries a netpositive charge and is typified by extracellular matrix components suchas collagen and laminin. Examples of commercially availableextracellular matrix components include Matrigel™ and Vitrogen™. Thethird class is net neutral in charge. An example of a net neutralhydrogel is highly crosslinked polyethylene oxide, or polyvinyalcohol.

Cores made of a hydrogel matrix are particularly suitable formaintaining cells or tissues which tend to form agglomerates oraggregates, such as the cells in islets of Langerhans, or adrenalchromaffin cells. The matrix should be of sufficient viscosity tomaintain cell dispersion within the matrix. Optionally, the core of theinstant vehicle can contain substances which support or promote thefunction of the isolated cells. These substances include natural orsynthetic nutrient sources, extracellular matrix (ECM) components,growth factors or growth regulatory substances, or a population offeeder or accessory cells or O₂ carriers such as hemoglobins andfluorocarbons.

Herein, the term “aggregating” refers to a process of promoting cellclustering. The cells which form clusters may be obtained from naturallyoccurring agglomerates, such as pancreatic islets which are dispersedinto single or small-clump suspension and subsequently reaggregatedaccording to the methods described. Alternatively, the cells may beobtained originally as single cells or small cell clumps, and thenaggregated to form the desired cluster size. Such cell clusters willgenerally contain about 3-400 cells, depending upon cell size andaggregation characteristics. Typically, the cluster contains about 10 toabout 50 cells. The use of reaggregated pancreatic islet cells isadvantageous for insuring proper diffusion characteristics within thecore and maintaining islet viability.

Reaggregated islets also allow the use of smaller capsules. Forinstance, 500 non-reaggregated islets would generally require a capsuleof approximately 14 cm in length (2% density). In contrast, capsulescontaining islets reaggregated to a size smaller than an intact isletmay be as small as 1 to 2 cm in length because of more efficientpacking. More efficient packing allows a lower pO₂ outside the fiber tobe tolerated without resultant necrotic cores. Built in tolerance forlower outside pO₂ has at least two advantages. Firstly, a smallercapsule size may be used to contain the same number of cells, i.e.increased tissue density within the implant is better tolerated.Secondly, implantation sites with known reduced pO₂, such assubcutaneous locations, may be used successfully. The presence of thealginate matrix further insures that the aggregates will not reassociateto large cell masses where internal cells would be deprived of nutrientsand/or oxygen.

In one advantageous application of the invention, the cells are formedby reaggregating natural cell clusters into a form adapted for increasedpacking per unit volume as described supra.

Such reaggregated clusters are preferably characterized by improveddiffusion of critical solutes to and from the cells within the clusterin comparison to natural cell clusters.

Cells which do not require an anchorage substrate are those which areable to form clumps or agglomerates and thus provide anchorage for eachother. Exemplary clumping cell types are pancreatic islets, pancreaticbeta-cell lines, Chinese hamster ovary (CHO) cells, and adrenalchromaffin cells. These cells are suitably enclosed in a negativelycharged matrix such as alginate.

Fibroblasts generally survive well in a positively charged matrix andare thus suitably enclosed in extracellular-matrix type hydrogels.Certain cell types tend to multiply rapidly and could overgrow the spaceavailable within the core if they do not exhibit growth arrest. If theisolated cells do not exhibit growth arrest upon confluence, substanceswhich induce quiescence can be included in the interior of the vehicle.In some instances, a hydrogel core may suffice to limit continuedproliferation. For example, a hydrogel matrix precursor solution can beincluded but not exposed to polymerizing conditions. In the case ofsodium alginate, a hydrogel will form slowly after implantation ascalcium ions are scavenged from the surrounding tissues. Alternatively,growth inhibitory factors, or stimulators of differentiation can beincorporated into microbeads of a slowly degraded polymer such aspolycarbonate, and cosuspended with the product-secreting cells. Forinstance, NGF or FGF can be used to stimulate PC-12 cell differentiationand terminate cell division.

Other cells, particularly primary cells or tissues, tend to adhere toeach other and form dense agglomerations which develop central necroticregions due to the relative inaccessibility of nutrients and oxygen tocells embedded within the agglomerated masses. These dense cellularmasses can form slowly, as a result of cell growth into dense colonies,or rapidly, upon the reassociation of freshly-dispersed cells or tissuemediated by cell-surface adhesion proteins. Cells or tissues which arehighly metabolically active are particularly susceptible to the effectsof oxygen or nutrient deprivation, and die shortly after becomingembedded in the center of an agglomerate. Many endocrine tissues, whichnormally are sustained by dense capillary beds, exhibit this behavior;islets of Langerhans and adrenal chromaffin cells are particularlysensitive. Cells or tissues which exhibit this behavior perform mostsatisfactorily in vehicles comprising a hydrogel matrix core, sufficientto immobilize the cells or tissue, thereby preserving the access ofnutrients and oxygen to the majority of them.

In other circumstances, the immobilizing hydrogel matrix furtherperforms the additional function of producing or preserving functionalunits of a size and/or shape appropriate for maintaining desirablecharacteristics of the isolated cells. Moreover, the presence of thecore matrix allows maintenance of a uniform distribution of cells orclusters of cells within the vehicle (i.e., the core matrix preventssettling and decreases mobility of the included cells).

One particularly advantageous use of hydrogel cores pertains to theencapsulation of actively dividing cells. Alginate or other hydrogelsmay be included in suspensions of actively dividing cells to beencapsulated. Following encapsulation and generation of the gel, theencapsulated cells are somewhat immobilized within the gel and new cellsproduced during cell division stay localized near the parent cell. Inthis manner clusters of cells are produced within the core. Such agrowth method is advantageous in the case of cells such as the beta cellderived NIT cell line. In the absence of a core matrix these cells tendto grow attached along the inner walls of the encapsulation device, withvery few cells growing freely within the cavity of the device. Growthonly on the walls of the capsule leads to a cell population size that isrestricted by the surface area of the inner capsule wall as opposed to apopulation that grows to fill the vehicle cavity. When alginate isincluded in the core, cell growth is no longer limited to the innercapsule surface. Rather, discrete spheres of NIT cells are producedthroughout the core, resulting in a significantly larger total cellpopulation than that which occurs in the absence of alginate.

Even in the presence of a core matrix, the size of tissue fragmentswhich can be loaded into a given vehicle volume is limited by theappearance of central necrosis within the individual fragments. In oneaspect of the instant invention, the useful amount of tissue fragmentsor cell clusters that can be placed within the immunoisolatory vehicleis increased by preparing the cells in a form with improved diffusionalcharacteristics. Generally this means preparation of the tissuefragments to a size less than 75 μm diameter and most optimally to about35 μm diameter or less for vehicles to be implanted peritoneally.Aggregates or clusters of cells in improved diffusional form areprepared for spontaneously reaggregating cells (e.g. pancreatic isletsor adrenal chromaffin cells) by enzymatically dispersing the tissue, tosingle cell and small cell aggregate suspensions, followed by controlledreaggregation to the improved diffusional form.

Pancreatic islet cells still retain functionality and secrete insulin inresponse to glucose in near normal fashion following enzymaticdispersion and reaggregation. Cells from dispersed islets arereaggregated to the desired cluster size prior to loading into thevehicle. Reaggregation can be accomplished by the methods described byBritt, Diabetes, 34, pp. 898-903, or by similar methods. The optimalaggregate size for islets is the smallest size cluster which stillmaintains the desired physiological characteristics. The matrix ormatrix forming materials may then be added to the cells, and thecombination may be coextruded into or loaded into biocompatibleimmunoisolatory vehicles. If necessary, matrix formation may then beinduced. In preferred. embodiments, cells are reaggregated overnight at37° C., without agitation. The development of aggregates is monitored bylight microscopy until aggregates achieve a size of 25 to 75 μmpreferably 35 μm diameter. Liquid uncrosslinked alginate is then addedto the cells to a concentration of from 0.5 to 2%, the cells areincorporated into vehicles, the vehicles are sealed if necessary, andpolymerization is induced by immersion of the vehicle in an aqueoussolution containing CaCl₂.

Primary cells or tissues may be useful with the vehicle of the instantinvention for various medical applications. For regulatory reasons andreasons of patient safety, it may sometimes be useful to employ assources for primary cultures, animals of carefully controlled hereditaryand developmental background. The presence of unwanted virus, bacteriaand other pathogens may be limited through the use of specific pathogenfree or gnotobiotic source animals. References and methods for theestablishment, care and use of specific pathogen free and gnotobioticherds are provided by Maniatis, O. P., et al. Can. J. Med., 42, p. 428(1978); Matthews P. J., et al., Recent Advances in Germ Free Research,pp. 61-64, Tokai Univ. Press (1981), and in the National AccreditationStandards publication of the National SPF Swine Accrediting Agency,Inc., Conrad, Iowa.

Optionally, a matrix core can also contain materials which support orpromote the function of the isolated cells. For example, extracellularmatrix (ECM) components can be included to promote specific attachmentor adhesion of the isolated cells. A combination of ECM components whichis particularly suitable for fostering the growth of certain types ofcells is taught in Kleinman et al., U.S. Pat. No. 4,829,000. The corematrix can provide a reservoir for soluble or releasable substances,such as growth factors or growth regulatory substances, or for naturalor synthetic substances which enhance or improve the supply of nutrientsor oxygen to the isolated cells. Thus, it can function in a mannersimilar to the bone marrow ECM, which has been reported to behave as aslow-release reservoir for myeloid lineage-specific growth factors suchas granulocyte-macrophage colony stimulating factor (gmcsf). Gordon, M.Y., et al., Nature, 326, pp. 403-405 (1987). Thus, the core matrix canfunction as a reservoir for growth factors (e.g., prolactin, orinsulin-like growth factor 2), growth regulatory substances such astransforming growth factor β (TGFβ) or the retinoblastoma gene proteinor nutrient-transport enhancers (e.g., perfluorocarbons, which canenhance the concentration of dissolved oxygen in the core). Certain ofthese substances are also appropriate for inclusion in liquid media.

Additionally, a population of feeder or accessory cells can becoisolated within the vehicle. For example, hepatocytes can becoisolated with endothelial accessory cells, Cai, Z., et al., ArtificialOrgans, 12, pp. 388-393 (1988), or mixed with islet cells, Ricordi C.,et al., Transplantation 45, pp. 1148-1151 (1987), or adrenal chromaffincells can be coisolated with accessory cells which provide nerve growthfactor (NGF), a substance needed by the chromaffin cells. In the lattercase, fibroblasts which have been transfected with an expression vectorfor NGF can be used as accessory cells.

The instant vehicle can also be used as a reservoir for the controlleddelivery of needed drugs or biotherapeutics. In such cases, the core,rather than containing cells or tissues, contains a high concentrationof the selected drug or biotherapeutic. In addition, satellite vehiclescontaining substances which prepare or create a hospitable environmentin the area of the body in which a biocompatible immunoisolatory vehiclecontaining isolated cells is implanted can also be implanted into arecipient. In such instances, the vehicle containing immunoisolatedcells is implanted in the region along with satellite vehicles releasingcontrolled amounts of, for example, a substance which down-modulates orinhibits an inflammatory response from the recipient (e.g.,anti-inflammatory steroids), or a substance which stimulates theingrowth of capillary beds (i.e., an angiogenic factor).

The surrounding or peripheral region (jacket) of the instant vehicle ispermselective, biocompatible, and immunoisolatory. It is produced insuch a manner that it is free of isolated cells, and completelysurrounds (i.e., isolates) the core, thereby preventing contact betweenany cells in the core and the recipient's body.

To be permselective, the jacket is formed in such a manner that it has aMWCO range appropriate both to the type and extent of immunologicalreaction it is anticipated will be encountered after the vehicle isimplanted and to the molecular size of the largest. substance whosepassage into and out of the vehicle is desirable. The type and extent ofimmunological attacks which may be mounted by the recipient followingimplantation of the vehicle depend in part upon the type(s) of moietyisolated within it and in part upon the identity of the recipient (i.e.,how closely the recipient is genetically related to the source of thebiologically active moiety). When the implanted tissue is allogeneic tothe recipient, immunological rejection may proceed largely throughcell-mediated attack by the recipient's immune cells against theimplanted cells. When the tissue is xenogeneic to the recipient,molecular attack through assembly of the recipient's cytolyticcomplement attack complex may predominate, as well as the antibodyinteraction with complement.

In reference to the permselectivity of the membranes and gels of theinstant invention, the phrase “molecular weight cutoff” (MWCO) is used.It is recognized that there are many methods available for thedetermination of the molecular weight cutoff for a permselectivemembrane. Depending upon the method used, somewhat different MWCOestimates may be achieved for the same membrane. In the context of thecurrent invention, MWCO refers to the results of the empiricaldeterminations described herein, using the specific markers describedunder the specific conditions of the determination. Other methods ofMWCO determination, to apply to the current invention, will need to becalibrated against the protocol of the instant invention according tomethods known to practitioners in the art.

The jacket allows passage of substances up to a predetermined size, butprevents the passage of larger substances. More specifically, thesurrounding or peripheral region is produced in such a manner that ithas pores or voids of a predetermined range of sizes; as a result, thevehicle is permselective. The molecular weight cutoff (MWCO) selectedfor a particular vehicle will be determined in part by the type andextent of immunological rejection it is anticipated will be encounteredafter the vehicle is implanted and in part by the molecular size of thelargest substance to be allowed to pass into and/or out of the vehicle.For example, materials can be used to form permselective membranes orhydrogel matrices which allow passage of molecules up to about the sizeof C1q, a component of complement (about 400 kD), a protein required forthe assembly of the cytolytic complement attack complex. In thisinstance, substances smaller than C1q can pass freely. It is alsopossible to form permselective matrices or membranes which allow passageof molecules up to about the size of immunoglobulin G (about 150 kD) andexclude larger molecules. Further, membranes or hydrogels which allowpassage of molecules up to about the size of immunoglobulin M (about1,000 kD) can be used; only very large substances, such as cells, willbe excluded in this embodiment.

The MWCO of the surrounding or peripheral region must therefore besufficiently low to prevent access of the substances required to carryout these attacks to the core, yet sufficiently high to allow deliveryof the needed product to the recipient's body. It will therefore beapparent that the MWCO need not be strictly restricted to a range whichexcludes immunoglobulin G from the core. In fact, there are many casesin which higher MWCOs are not only permissible, but also advantageous.Indeed, higher MWCOs allow the delivery of a wide variety of usefulproducts from immunoisolated cells, as well as the use of such cells toprovide metabolic control of high molecular weight substances.

Thus, in appropriate cases, the peripheral or surrounding region can bemade of materials which form permselective membranes or hydrogelmatrices allowing the passage of molecules up to about the size of C1q(about 400 kD), the largest protein required for the assembly of thecomplement attack complex. Therefore, any cellular product or metabolitebelow about the size of C1q can pass freely through the vehicle. Inother cases, it may still be desirable to exclude immuno-globulins. Insuch cases, materials which form matrices or membranes through whichmolecules which are equivalent to or larger than the size ofimmunoglobulin G (about 150 kD) cannot pass can be used. Cellularproducts or metabolites which are smaller than about 150 kD will stillpass through the vehicle. In still other cases, where the patient isimmunosuppressed or where the implanted tissue is syngeneic to thepatient, a vigorous immunological attack is not likely to beencountered, and passage of a high molecular weight molecule may bedesired. In these latter cases, materials which allow passage of allmolecules up to about the size of immunoglobulin M (about 1,000 kD) canbe used. These materials will impede the passage of only very largesubstances, such as cells.

In another aspect of the invention, it has been found that a molecularweight cutoff for the jacket considerably higher than that previouslycontemplated may be employed while maintaining the viability andfunction of the encapsulated cells. This permits the macrocapsules to beused in applications where the cells secrete a substance of highmolecular weight. For this purpose, a macrocapsule with molecularcutoffs in excess of say 80 to 100 kD to as high as 200 to 1000 or 2000kD or more may be employed in accordance with the present invention.

As used herein, the term “biocompatible” refers collectively to both theintact vehicle and its contents. Specifically, it refers to thecapability of the implanted intact vehicle and its contents to avoiddetrimental effects of the body's various protective systems and remainfunctional for a significant period of time. In addition to theavoidance of protective responses from the immune system, or foreignbody fibrotic response, “biocompatible” also implies that no specificundesirable cytotoxic or systemic effects are caused by the vehicle andits contents such as would interfere with the desired functioning of thevehicle or its contents.

The jacket is biocompatible. That is, it does not elicit a detrimentalhost response sufficient to result in rejection of the implanted vehicleor to render it inoperable. Neither does the jacket elicit unfavorabletissue responses such as fibrosis. In addition, the external surface canbe selected or designed in such a manner that it is particularlysuitable for implantation at the selected site. For example, theexternal surface can be smooth, stippled or rough, depending on whetherattachment by dells of the surrounding tissue is desirable. The shape orconfiguration can also be selected or designed to be particularlyappropriate for the implantation site chosen.

The biocompatibility of the surrounding or peripheral region (jacket) isproduced by a combination of factors.

Important for biocompatibility and continued functionality are vehiclemorphology, hydrophobicity and the absence of undesirable substanceseither on the surface of, or leachable from, the vehicle itself. Thus,brush surfaces, folds, interlayers or other shapes or structureseliciting a foreign body response are avoided. The vehicle-formingmaterials are sufficiently pure that unwanted substances do not leachout from the vehicle materials themselves. Additionally, followingvehicle preparation, the treatment of the external surface of thevehicle with fluids or materials (e.g. serum) which may adhere to or beabsorbed by the vehicle and subsequently impair vehicle biocompatibilityare avoided.

First, the materials used to form the vehicle are substances selectedbased upon their ability to be compatible with, and accepted by, thetissues of the recipient of the implanted vehicle. Substances are usedwhich are not harmful to the recipient or to the isolated biologicallyactive moiety. Preferred substances include reversibly and irreversiblygellable substances (e.g., those which form hydrogels), andwater-insoluble thermoplastic polymers. Particularly preferredthermoplastic polymer substances are those which are modestlyhydrophobic ,i.e. those having a solubility parameter as defined inBrandrup J., et al. Polymer Handbook 3rd Ed., John Wiley & Sons, N.Y.(1989), between 8 and 15, or more preferably, between 9 and 14(Joules/m³)^(1/2). The polymer substances are chosen to have asolubility parameter low enough so that they are soluble in organicsolvents and still high enough so that they will partition to form aproper membrane. Such polymer substances should be substantially free oflabile nucleophilic moieties and be highly resistant to oxidants andenzymes even in the absence of stabilizing agents. The period ofresidence in vivo which is contemplated for the particularimmunoisolatory vehicle must also be considered: substances must bechosen which are adequately stable when exposed to physiologicalconditions and stresses. There are many hydrogels and thermoplasticswhich are sufficiently stable, even for extended periods of residence invivo, such as periods in excess of one or two years. Examples of stablematerials include alginate (hydrogel) andpolyacrilonitrile/polyvinylchloride (“PAN/PVC” or “thermoplastic”).

Second, substances used in preparing the biocompatible immunoisolatoryvehicle are either free of leachable pyrogenic or otherwise harmful,irritating, or immunogenic substances or are exhaustively purified toremove such harmful substances. Thereafter, and throughout themanufacture and maintenance of the vehicle prior to implantation, greatcare is taken to prevent the adulteration or contamination of thevehicle with substances which would adversely affect itsbiocompatibility.

Third, the exterior configuration of the vehicle, including its texture,is formed in such a manner that it provides an optimal interface withthe tissues of the recipient after implantation. This parameter will bedefined in part by the site of implantation. For example, if the vehiclewill reside in the peritoneal cavity of the recipient, its surfaceshould be smooth. However, if it will be embedded in the soft tissues ofthe recipient, its surface can be moderately rough or stippled. Adetermining factor will be whether it is desirable to allow cells of therecipient to attach to the external surface of the vehicle or if suchattachment must be avoided. An open-textured or sponge-like surface maypromote the ingrowth of capillary beds, whereas a smooth surface maydiscourage excessive overgrowth by fibroblasts. Excessive overgrowth byfibroblasts is to be avoided, except where capillary undergrowth hasoccurred, as it may result in the deposition of a poorly-permeablebasement membrane around the vehicle and walling off of the isolatedcells from contact with the recipient's body.

Certain vehicle geometries have also been found to specifically elicitforeign body fibrotic responses and should be avoided. Thus vehiclesshould not contain structures having interlayers such as brush surfacesor folds. In general, opposing vehicle surfaces or edges either from thesame or adjacent vehicles should be at least 1 mm apart, preferablygreater than 2 mm and most preferably greater than 5 mm. Preferredembodiments include cylinders, “U”-shaped cylinders, and flat sheets orsandwiches.

The surrounding or peripheral region (jacket) of the biocompatibleimmunoisolatory vehicle can optionally include substances which decreaseor deter local inflammatory response to the implanted vehicle, and/orgenerate or foster a suitable local environment for the implanted cellsor tissues. For example antibodies to one or more mediators of theimmune response could be included. Available potentially usefulantibodies such as antibodies to the lymphokines tumor necrosis factor(TNF), and to interferons (IFN) can be included in the matrix precursorsolution. Similarly, an anti-inflammatory steroid can be included.Christenson, L., et al., J. Biomed. Mat. Res., 23, pp. 705-718 (1989);Christenson, L., Ph.D. thesis, Brown University, 1989, incorporated byreference.

Alternatively, a substance which stimulates angiogenesis (ingrowth ofcapillary beds) can be included. This may be particularly desirablewhere the isolated cells or tissues require close contact with therecipient's bloodstream to function properly (e.g., insulin-producingislets of Langerhans). Cell which are genetically engineered to secreteantibodies may also be included in the matrix.

Because of its biocompatibility, the vehicle is suitable for long-termisolation of biologically useful cells and/or substances from thevarious protective systems of the body. As used herein, the term“protective systems” refers to the types of immunological attack whichcan be mounted by the immune system of an individual in whom the instantvehicle is implanted, and to other rejection mechanisms, such as thefibrotic response, foreign body response and other types of inflammatoryresponse which can be induced by the presence of a foreign object in theindividuals' body.

The jacket of the present vehicle is immunoisolatory. That is, itprotects cells in the core of the vehicle from the immune system of theindividual in whom the vehicle is implanted. It does so (1) bypreventing harmful substances of the individual's body from entering thecore of the vehicle, (2) by minimizing contact between the individualand inflammatory, antigenic, or otherwise harmful materials which may bepresent in the core and (3) by providing a spatial and physical barriersufficient to prevent immunological contact between the isolated moietyand detrimental portions of the individual's immune system.

The thickness of this physical barrier can vary, but it will always besufficiently thick to prevent direct contact between the cells and/orsubstances on either side of the barrier. The thickness of this regiongenerally ranges between 5 and 200 microns; thicknesses of 10 to 100microns are preferred, and thickness of 20 to 50 microns areparticularly preferred. Types of immunological attack which can beprevented or minimized by the use of the instant vehicle include attackby macrophages, neutrophils, cellular immune responses (e.g. naturalkiller cells and antibody-dependent T cell-mediated cytoloysis [ADCC]),and humoral response (e.g. antibody-dependent complement mediatedcytolysis).

The type and extent of immunological response by the recipient to theimplanted vehicle will be influenced by the relationship of therecipient to the isolated biologically active moiety. For example, ifthe isolated materials comprise syngeneic cells, these will not cause avigorous immunological reaction, unless the recipient suffers from anautoimmunity with respect to the particular cell or tissue type withinthe vehicle. There are several disease or deficiency states which haverecently been determined to have an autoimmune etiology, most notablyType I, insulin-dependent Diabetes mellitus, wherein the insulinsecreting pancreatic islet β cells are destroyed by the individual'simmune system. Fan, M.-Y. et al., Diabetes, 39, pp. 519-522 (1990).

Syngeneic cells or tissue are rarely available. In many cases,allogeneic or xenogeneic cells or tissue (i.e., from donors of the samespecies as, or from a different species than, the prospective recipient)may be available. The immunoisolatory vehicle allows the implantation ofsuch cells or tissue, without a concomitant need to immunosuppress therecipient. Therefore, the instant vehicle makes it possible to treatmany more individuals than can be treated by conventionaltransplantation techniques. For example, far more patients suffer fromType 1 diabetes than can be transplanted with human donor islets (in1990, fewer than about 4,000 suitable cadaver organ donors becameavailable in the U.S. for all organ transplants). The supply of donorporcine or bovine islets is far greater; if these xenoislets areappropriately immunoisolated according to the instant invention, thediabetic condition of a far greater number of patients can be remedied.

The type and vigor of an immune response to xenografted tissue isexpected to differ from the response encountered when syngeneic orallogeneic tissue is implanted into a recipient. This rejection mayproceed primarily by cell-mediated, or by complement-mediated attack;the determining parameters in a particular case may be poorlyunderstood. However, as noted previously, the exclusion of IgG from thecore of the vehicle is not the touchstone of immunoprotection, becauseIgG alone is insufficient to produce cytolysis of the target cells ortissues.

Using the macrocapsules of the present invention, preferably withallogeneic tissue, but even with xenografts, it is possible to deliverneeded high molecular weight products or to provide metabolic functionspertaining to high molecular weight substances, provided that criticalsubstances necessary to the mediation of immunological attack areexcluded from the immunoisolatory vehicle. These substances may comprisethe complement attack complex component C1q, or they may comprisephagocytic or cytotoxic cells; the instant immunoisolatory vehicleprovides a protective barrier between these harmful substances and theisolated cells. Thus, the present immunoisolatory vehicle can be usedfor the delivery even from allogeneic or xenogeneic cells or tissue,products having a wide range of molecular sizes, such as insulin,parathyroid hormone, interleukin 3, erythropoietin, albumin,transferrin, and Factor VIII.

The jacket of the instant vehicle is made of a material which may be thesame as that of the core or may be different. In either case, thematerial used results in a surrounding or peripheral region which ispermselective, biocompatible and immunoisolatory. The jacket may beformed freely around the core without chemical bonding or,alternatively, the jacket may be directly cross-linked to the corematrix. In either case, formation of the vehicle of the presentinvention does not require polymers of opposite charge to the core beingpresent in an interfacial layer or in the jacket.

The surrounding or peripheral region (jacket) can be made of a hydrogelmatrix or of a different material, such as a thermoplastic membrane. Itcan also be made of a matrix-membrane composite, such that apermselective thermoplastic membrane having matrix-filled pores, isformed.

Suitably, the external jacket may be formed of a thermoplastic materialknown to be biocompatible, such as the ones described herein. Inaddition, other jackets which have been used in the microcapsule fieldmay also be used herein, such as alginate, suitably cross-linked with amultivalent ion such as calcium.

Preferably, the core and external jacket form an interface free of“ionic bonding” between oppositely charged polymers and free of anintermediate layer of the type used in prior art microcapsules. Ionicbonding refers to an ionic interaction of a core of one charge (positiveor negative) and the jacket (or an intermediate layer) of oppositecharge.

In previously existing devices, the core and jacket were linked viaionic bonds between oppositely charged polymers in one of two ways. (1)The devices of Rha (U.S. Pat. No; 4,744,933) were constructed of acharged inner core material and an outer jacket material of the oppositecharge. (2) The devices of Lim and Sun (U.S. Pat. Nos. 4,352,833 and4,409,331) included an intermediate layer of poly-L-lysine (PLL), whichis positively charged, to link the negatively charged core with thenegatively charged jacket material. The elimination of a PLL layer isadvantageous in that PLL is believed to be fibrogenic in the host. PLLmay also have unwanted growth effects for some cells. Also, the jacketof the device of the invention can be controlled for permselectivitybetter than those made with PLL.

The vehicle of the present invention is distinguished from themicrocapsules of Lim and Sun (Lim, F., Sun, A. M., Science 210, pp.908-910 (1980); Sun, A. M., Methods in Enzymology 137, pp. 575-579(1988) by its outer jacket which ensures that cells cannot projectoutside of the core. The capsules of Lim and Sun suffered from thedisadvantage that portions of encapsulated cells could potentiallyproject from the core through the poly-L-lysine layer and thereby bemore likely to elicit inflammatory responses from the host's immunesystem. That microcapsule technology relies on the presence ofpotentially bioactive ionic bonds to form the microcapsule. By virtue oftheir ionic nature, those microcapsules are susceptible to deteriorationfollowing implantation due to competition for the ionic bonds that takeplace in the body of the host after capsule implantation. This problemis minimized by the relatively non-ionic macrocapsules of the presentinvention. A further advantage of the macrocapsules of the presentinvention lies in their capacity to contain more cells in a singledevice than is possible in microcapsule technology.

The term “dual matrix vehicles” refers to vehicles with acell-containing core and an external jacket free of cells. In oneembodiment, the matrix core is formed of a hydrogel which iscross-linked to a hydrogel jacket, suitably in the form of a rod orother shape. The hydrogel jacket may be formed independently as a sheatharound the matrix without cross-linking. The hydrogel care is notnecessarily linked to the outer jacket by means of opposite ioniccharges. In another embodiment, the external jacket is formed of athermoplastic material which is not linked to the core matrix bychemical bonding.

If a dual matrix immunoisolatory vehicle is to be formed, thesurrounding or peripheral region can be made of a hydrogel selected fromthe above-listed matrix precursors. If the surrounding or peripheralregion of the vehicle is to comprise a permselective membrane, otherprecursor materials can be used. For example, the surrounding orperipheral region can be made from water-insoluble, biocompatiblethermoplastic polymers or copolymers. Several of the polymers orcopolymers taught by Michaels, U.S. Pat. No. 3,615,024, which is herebyincorporated by reference, fulfill these criteria.

A preferred membrane casting solution comprises apolyacrylonitrile-polyvinylchloride (PAN/PVC) copolymer dissolved in thewater-miscible solvent dimethylsulfoxide (DMSO). This casting solutioncan optionally comprise hydrophilic or hydrophobic additives whichaffect the permeability characteristics of the finished membrane. Apreferred hydrophilic additive for the PAN/PVC copolymer ispolyvinylpyrrolidone (PVP). Other suitable polymers comprisepolyacrylonitrile (PAN), polymethyl-methacrylate (PMMA),polyvinyldifluoride (PVDF), polyethylene oxide, polyolefins (e.g.,polyisobutylene or polypropylene), polysulfones, and cellulosederivatives (e.g., cellulose acetate or cellulose butyrate). Compatiblewater-miscible solvents for these and other suitable polymers andcopolymers are found in the teachings of U.S. Pat. No. 3,615,024.

In a preferred embodiment, the core is surrounded by a biocompatiblehydrogel matrix free of cells projecting externally from the outerlayer. The macrocapsules of the present invention are distinguished fromthe microcapsules of Rha, Lim, and Sun (Rha, C. K. et al., U.S. Pat. No.4,744,933; Sun, A. W., supra) by (1) the complete exclusion of cellsfrom the outer layer of the macrocapsule, and (2) the thickness of theouter layer of the macrocapsule. Both qualities contribute to theimmunoisolation of encapsulated cells in the present invention. Themicrocapsules of Rha were formed by ionic interaction of an ionic coresolution with an ionic polymer of opposite charge. The microcapsules ofLim and Sun were formed by linking an external hydrogel jacket to thecore through an intermediate layer of poly-L-lysine (PLL).

In the microcapsules of Lim and Sun, the intermediate PLL layer was notsufficiently thick to guarantee that portions of the encapsulated cellswould not penetrate through and beyond the layer. Cells penetrating thePLL layer are potential targets for an immune response. All thesecapsules, including those disclosed by Rha, also suffer the followingadditional limitations: (a) they are round, and (b) the formation of theouter layer is dependent upon direct ionic bonding or polyamide linkagewith an inner layer or core substance. The disadvantages of round shapeand direct ionic bonding between polymers are described supra.

In the capsules of Rha, Lim, and Sun, since the chemical identity of theinner substance is either dictated by choice of outer layer, or PLL, theability to vary growth conditions on the inside of these capsules isgreatly limited. Since there are often specific growth conditions whichneed to be met in order to successfully encapsulate specific cell types,these capsules generally have a limited utility or require considerableexperimentation to establish appropriate outer layers for a giveninternal substance. In contrast, in the instant invention, the identityof the core material does not place strict limitations on the identityof the outer jacket material or vice versa. This allows the material ofthe inner hydrogel to be selected according to criteria important forcell viability and growth, and the outer jacket material to be selectedon the basis of immunoisolatory properties, biocompatibility, and/ormanufacturing considerations.

The microcapsules of Rha, Lim, and Sun have a greater potential forbioincompatibility, fibrogenesis, and vehicle deterioration than do themacrocapsules of the present invention. A variety of biological systemsare known to interact with and break down the ionic bonds required forthe integrity of microcapsules. PLL evokes unfavorable tissue reactionsto the capsule. Most notably, this is a fibrotic response. Thus, ifthere is any break in the external layer, if it is not of sufficientthickness, if the PLL layer begins to degrade, or if encapsulated cellsare entrapped within the external layer sufficiently close to its outersurface, the microcapsule can trigger a fibrotic response. The term“fibrogenic” is used herein in reference to capsules or materials whichelicit a fibrotic response in the implantation site. As set forthherein, the external jacket of the immunoisolatory, non-fibrogenicmacrocapsule of the present invention may be formed in a number of ways.

In one embodiment, the core is preformed by cross-linking a hydrogelmatrix with a cross-linking agent, preferably a multivalent cation suchas calcium. However, other known hydrogel cross-linking agents may alsobe employed. After cross-linking, the core is dipped into a solution ofhydrogel to form a second layer free of cells in the core which,simultaneously or thereafter, is cross-linked suitably in the samemanner. In the instant embodiment, cross-linking of the core materialwith the jacket material is accomplished via the cross-linking agent.For instance, when the core and jacket materials are both negativelycharged hydrogels, the core and the jacket are cross-linked with eachother via their mutual attraction to the positive charges on thecross-linking agent, preferably calcium. The core and jacket may beformed of the same or different type of hydrogel, provided that bothhave the same charge. Notably, the instant vehicle is not formed throughdirect ionic bonding between anionic and cationic polymers as describedin Rha, C. K., U.S. Pat. No. 4,744,933.

Herein, the term “direct ionic bonding” refers to the type of chemicalbonding in which two oppositely charged polymers are attracted to oneanother because of their oppositely charged moieties. The instantembodiment is distinguished from that of Rha because, in the instantembodiment, both the core material and jacket material have the samecharge, and their association is via an oppositely charged cross-linkingagent. This embodiment may be in the form of a microcapsule or amacrocapsule but, for reasons set forth herein, the macrocapsule form ispreferred.

The present immunoisolatory vehicle can be formed in a wide variety ofshapes and combinations of suitable materials. A primary considerationin selecting a particular configuration for the vehicle when cells arepresent is the access of oxygen and nutrients to the isolated cells ortissues, and passage of waste metabolites, toxins and the secretedproduct from the vehicle. The immunoisolatory vehicle can be anyconfiguration appropriate for maintaining biological activity andproviding access for delivery of the product or function, including forexample, cylindrical, rectangular, disk-shaped, patch-shaped, ovoid,stellate, or spherical. Moreover, the vehicle can be coiled or wrappedinto a mesh-like or nested structure. If the vehicle is to be retrievedafter it is implanted, configurations which tend to lead to migration ofthe vehicle(s) from the site of implantation, such as spherical vehiclessmall enough to travel in the recipient's blood vessels, are notpreferred. Certain shapes, such as rectangles, patches, disks,cylinders, and flat sheets offer greater structural integrity and arepreferable where retrieval is desired.

The instant vehicle must provide, in at least one dimension,sufficiently close proximity of any isolated cells in the core to thesurrounding tissues of the recipient, including the recipient'sbloodstream, in order to maintain the viability and function of theisolated cells. However, the diffusional limitations of the materialsused to form the vehicle do not in all cases solely prescribe itsconfigurational limits. Certain additives can be used which alter orenhance the diffusional properties, or nutrient or oxygen transportproperties, of the basic vehicle. For example, the internal medium canbe supplemented with oxygen-saturated perfluorocarbons, thus reducingthe needs for immediate contact with blood-borne oxygen. This will allowisolated cells or tissues to remain viable while, for instance, agradient of angiotensin is released from the vehicle into thesurrounding tissues, stimulating ingrowth of capillaries. References andmethods for use of perfluorocarbons are given by Faithful, N. S.Anaesthesia, 42, pp. 234-242 (1987) and NASA Tech Briefs MSC-21480, U.S.Govt. Printing Office, Washington, D.C. 20402, incorporated herein byreference. Alternatively for clonal cell lines such as PC12 cells,genetically engineered hemoglobin sequences may be introduced into thecell lines to produce superior oxygen storage. NPO-17517 NASA TechBriefs, 15, p. 54.

In general, in the absence of oxygen carrier additives, when the cellsare present the vehicle will have a maximum depth-to-surface distance ofno more than 2 mm in at least one dimension, with a maximum depth of 800microns being preferred. One or several vehicles may be required toproduce the desired effect in the recipient.

The thickness of the immunoisolatory vehicle jacket should be sufficientto prevent an immunoresponse by the patient to the presence of thevehicles. For that purpose, the vehicles preferably have a minimumthickness of 1 μm or more free of the cells.

Additionally, reinforcing structural elements can be incorporated intothe vehicle. These structural elements can be made in such a fashionthat they are impermeable, and are appropriately configured to allowtethering or suturing of the vehicle to the tissues of the recipient. Incertain circumstances, these elements can act to securely seal thesurrounding or peripheral region (e.g., at the ends of a cylindricalvehicle, or at the edges of a disk-shaped vehicle), completing isolationof the core materials (e.g., a molded thermoplastic clip). For manyconfigurations, it is desirable that these structural elements shouldnot occlude a significant area of the permselective surrounding orperipheral region.

In one preferred embodiment, the implantable immunoisolatory vehicle ofthe present invention is of a sufficient size and durability forcomplete retrieval after implantation. To be contrasted with suchmicrocapsules, which have a typical maximum practical volume on theorder of 1 μl, the preferred immunoisolatory vehicle of the presentinvention is termed “macrocapsule”. Such macrocapsules have a core of apreferable minimum volume of about 1 to 10 μl and depending upon use areeasily fabricated to have a value in excess of 100 μl.

In terms of retrievability, microspheres are generally less practicalthan macro-capsules. In order for tissue encapsulated in microspheres toprovide a therapeutic dose of insulin, for instance, the number ofmicrospheres must be increased to such a large extent that significantretrievability becomes impossible. Additionally, an increase in thevolume of tissue placed within a microsphere requires a correspondingincrease in surface area. Within a sphere, because surface area scaleswith r² where as volume scales with r³, as the volume of encapsulatedtissue volume increases, the required capsule size to provide sufficientsurface area for nutrient diffusion to the encapsulated tissue quicklybecomes impractical.

Macrocapsules in the shapes of cylinders or flat sheets do not havethese limitations because volume increases more proportionately tosurface area such that the diffusional transport of nutrients andproducts for increased amounts of tissue can be accommodated byincreasing the surface area without unwieldy increases in total vehiclesize. If, for example, about 10,000 islets are required per kg bodyweight to restore normoglycemia to a diabetic patient, from 1,000 to10,000 microcapsules must be transplanted per kg body weight (e.g. 1-10islets per capsule). This number of microcapsules could not be easilyretrieved, if retrieval were required. In contrast, the macrocapsules ofthe instant invention may easily hold greater than 1,000 islets to ashigh as 500,000 islets or more per vehicle. The preferred embodimentwould require fewer than 5-10 vehicles per patient, making macrocapsulesmore easily retrieved than a large number of microcapsules.

The macrocapsules of the present invention are distinguished frommicrocapsules (Sun, A. M., supra; Rha, U.S. Pat. No. 4,744,933) by thecapacity of macrocapsules to contain over 10⁴ cells and maintain them inviable condition. The droplet methods used in the making ofmicrocapsules, in order to ensure cell viability, necessarily limit thenumber of cells per capsule to fewer than 10⁴.

The instant invention also relates to a method for making animmunoisolatory vehicle. The vehicles of this invention can be formedeither by coextrusion or stepwise assembly. Techniques for coextrusionwhich can be used to form the instant vehicle are taught in copendingU.S. patent application Ser. No. 07/461,999, filed Jan. 8, 1990, whichis herein incorporated by reference. For example, a coextrusion devicesimilar to that taught by U.S. Ser. No. 07/461,999 is used in making thesubject vehicle. The device has a nested-bore extrusion nozzle, thelumen of each bore (inner and outer) being appropriately connected tosterile chambers for delivery of the core and surrounding regionmaterials.

The nozzle can be of any configuration appropriate to produce animmunoisolatory vehicle whose shape is appropriate to the metabolicneeds of the cells to be immobilized and the permeability and strengthof the matrix which will surround them. For example, the nozzle can becircular, elliptical, stellate, or slot-shaped. Optionally, the nestedbores can be coaxial. The widest aperture of the nozzle must becommensurate with the maximum diffusional depth appropriate to thevehicle being formed, including the metabolic needs of the isolatedcells or tissues, and the materials of the core and peripheral regions.

Upon extrusion of the core and peripheral region materials from theinner and outer chambers through the corresponding bores of the nozzleunder conditions sufficient to gel, harden, or cast the matrix ormembrane precursor(s) of the surrounding or peripheral region (and ofthe core region), an elongated vehicle of the selected shape iscontinuously formed. The length of the vehicle, and therefore its volumeor capacity, can be controlled to produce vehicles of sizes appropriatefor the particular use contemplated.

An immunoisolatory vehicle formed by coextrusion in this manner isparticularly preferred because use of this means ensures that the cellsin the core are isolated from the moment of formation of the vehicle.Thus, it also ensures that the core materials do not become contaminatedor adulterated during handling of the vehicle prior to implantation.

Furthermore, the nature of the coextrusion process is such that itensures that the surrounding or peripheral region (jacket) is free ofthe materials in the core, including the cells, thus assuring that thesecells will be immunoisolated when the vehicle is implanted into anindividual. The permeability, molecular weight cutoff, andbiocompatibility characteristics of the surrounding or peripheral regionare determined by both the chosen matrix or membrane precursor materialsused, and the conditions under which the matrix or membrane is formed.

The co-extruded vehicles may be formed with a hydrogel matrix core and athermoplastic or hydrogel jacket. Such macrocapsules may be formed witha core and jacket of the same or different hydrogel which may becross-linked to each other or not.

If a dual-matrix immunoisolatory vehicle (e.g., an alginate matrix) isformed, the permeability of the surrounding matrix can be determined byadjusting the concentration of matrix precursor used (e.g., sodiumalginate), and/or the concentration of gelling agent (in the case ofalginate, a divalent cation such as Ca⁺⁺) present in an immersion bathinto which the materials are coextruded.

If an immunoisolatory vehicle with a surrounding or peripheral region ofthermoplastic membrane is desired, the pore size range and distributioncan be determined by varying the solids content of the solution ofprecursor material (the casting solution), the chemical composition ofthe water-miscible solvent, or optionally including a hydrophilic orhydrophobic additive to the casting solution, as taught by U.S. Pat. No.3,615,024. The pore size may also be adjusted by varying thehydrophobicity of the coagulant and/or of the bath.

Typically, the casting solution will comprise a polar organic solventcontaining a dissolved, water-insoluble polymer or copolymer. Thispolymer or copolymer precipitates upon contact with a solvent-miscibleaqueous phase, forming a permselective membrane at the site ofinterface. The size of pores in the membrane depends upon the rate ofdiffusion of the aqueous phase into the solvent phase; the hydrophilicor hydrophobic additives affect pore size by altering this rate ofdiffusion. As the aqueous phase diffuses farther into the solvent, theremainder of the polymer or copolymer is precipitated to form atrabecular support which confers mechanical strength to the finishedvehicle.

The external surface of the vehicle is similarly determined by theconditions under which the dissolved polymer or copolymer isprecipitated (i.e., exposed to the air, which generates an open,trabecular or sponge-like outer skin, immersed in an aqueousprecipitation bath, which results in a smooth permselective membranebilayer, or exposed to air saturated with water vapor, which results inan intermediate structure). Again, it will be readily appreciated thatthis method of forming the immunoisolatory vehicle ensures that theperipheral or surrounding membrane is free of the cells in the corewhich are desired to be isolated from the immune system of theindividual in whom the vehicle is to be implanted.

The surface texture of the vehicle is dependent in part on whether theextrusion nozzle is positioned above, or immersed in, the bath: if thenozzle is placed above the surface of the bath a roughened outer skin ofPAN/PVC will be formed, whereas if the nozzle is immersed in the bath asmooth external surface is formed.

The immunoisolatory vehicle can also be formed in a stepwise manner. Forvehicles wherein the core comprises, in addition to the cells desired tobe isolated, a hydrogel matrix, the core can be formed initially, andthe surrounding or peripheral matrix or membrane can be assembled orapplied subsequently. The matrix core can either be formed by extrusionor by molding. In a preferred embodiment, a patch- or sheet-shapedvehicle is formed by stepwise extrusion of calendared sheets. In thisembodiment, a sheet of core material is layered onto a sheet ofperipheral region material, then covered by a second sheet of peripheralregion material. The edges of the vehicle are then sealed by crimping,compressing, heating, sealing with a biocompatible glue, or binding intoa preformed biocompatible impermeable clip or combinations of the above.

Conversely, the surrounding or peripheral matrix or membrane can bepreformed, filled with the materials which will form the core (forinstance, using a syringe), and subsequently sealed in such a mannerthat the core materials are completely enclosed. The vehicle can then beexposed to conditions which bring about the formation of a core matrixif a matrix precursor material is present in the core. Alternatively, apatch- or sheet-shaped matrix core can be formed by molding, thensandwiched between sheets of permselective membrane and sealed orclipped in the manner described above to complete the isolation of thecore materials.

It is also possible for a single, continuous hydrogel matrix to provideboth immunoisolation and support or immobilization. For example, thiscan be accomplished by including the isolated cells within the vehicledistributed in a concentric gradient about the core, such that theperipheral region of the vehicle is free of the immobilized cells.Immunoisolatory vehicles of this nature can be made in at least twoways. First, a mixture of cells suspended in a solution of a hydrogelmatrix precursor, which is denser than the isolated cells, can beextruded from the nozzle of a simple extrusion device. In this manner,the cells are forced into the core region of the forming vehicle.Alternatively, the cell-matrix precursor mixture can be extruded fromthe core lumen of a nested-bore nozzle, while simultaneously deliveringa stream of a gelling agent (e.g., for alginate, a solution of calciumchloride) through the peripheral nozzle, whereby the surface andperiphery of the vehicle polymerize first, thus forcing the suspendedcells into the core.

As noted previously, the vehicle can provide for the implantation ofdiverse cell or tissue types, including fully-differentiated,anchorage-dependent, fetal or neonatal, or transformed,anchorage-independent cells or tissue. The cells to be immunoisolatedare prepared either from a donor (i.e., primary cells or tissues,including adult, neonatal, and fetal cells or tissues) or from cellswhich replicate in vitro (i.e., immortalized cells or cell lines,including genetically modified cells). In all cases, a sufficientquantity of cells to produce effective levels of the needed product orto supply an effective level of the needed metabolic function isprepared, generally under sterile conditions, and maintainedappropriately (e.g. in a balanced salt solution such as Hank's salts, orin a nutrient medium, such as Ham's F12) prior to isolation.

In another aspect of the invention, the macrocapsules are of a shapewhich tends to reduce the distance between the center of themacrocapsule and the nearest portion of the jacket for purposes ofpermitting easy access of nutrients from the patient into the cell or ofentry of the patient's proteins into the cell to be acted upon by thecell to provide a metabolic function, such as interaction withcholesterol or the like. In that regard, a non-spherical shape ispreferred, such as a long tube or flat plate, or the like. The optimumshape for this purpose may be calculated by known techniques as setforth herein.

Four important factors influencing the number of cells or amount oftissue to be placed within the core of the biocompatible immunoisolatoryvehicle (i.e., loading density) of the instant invention are: (1)vehicle size and geometry; (2) mitotic activity within the vehicle; (3)viscosity requirements for core preparation and or loading; and (4)pre-implantation assay and qualification requirements.

With respect to the first of these factors, (capsule size and geometry),the diffusion of critical nutrients and metabolic requirements into thecells as well as diffusion of waste products away from the cell arecritical to the continued viability of the cells. Since diffusionalaccess to the contents of the vehicle is limited by vehicle surfacearea, surface to volume relationships of various shapes and sizevehicles will be critical in determining how much viable tissue can bemaintained within the vehicle.

Among the metabolic requirements met by diffusion of substances into thevehicle is the requirement for oxygen. The oxygen requirements of thespecific cells must be determined for the cell of choice. Methods andreferences for determination of oxygen metabolism are given in Wilson D.F. et al., J. Biol. Chem., 263, pp. 2712-2718, (1988). The oxygenrequirement for islet cells has been applied to coupled diffusionreaction models accounting for diffusional transport from surroundingtissue through the vehicle wall and tissue compartment (core), and usedto calculate the expected viability of islet cells in a number ofvehicles of different sizes and configurations, after the method ofDionne, K. E., Ph.D. Thesis, Massachusetts Institute of Technology(1989). For intact pancreatic islets, these calculations agree well withexperimental observations.

For a cylindrical vehicle of 900 microns outer diameter, implanted intothe peritoneal cavity (pO2≈45-50 mmHg), the optimal total cell volume isin th range of up to 20%, preferably 1-15%, most preferably about 5% ofthe vehicle volume. If this capsule were 20 cm in length it would have avolume of 100 mm³. To provide the same amount of surface area with asingle sphere, e.g. to support comparable amounts of tissue, wouldrequire a volume of 1,047 mm³.

For a cylindrical vehicle of 400 microns the optimal cell volume isbetween 35-65% total vehicle volume, and is preferably 35%. Thesecalculations also take into account the partial oxygen pressure at thesite of implantation. At implantation sites where the oxygen pressure isless than the peritoneum (e.g., subcutaneous pO2≈20 mmHg), lower loadingdensities will be required. Implantation into arteries (pO2>95 mmHg) andthe brain (pO2>75 mmHg) will allow support of greater tissue volume perunit vehicle.

Other vehicle configurations, such as disk-shaped or spherical, are alsopossible and optimal cell volumes may be similarly calculated for thesegeometries. Actual loading densities will consider not only thesediffusional considerations but also the other considerations givenbelow.

With respect to the second factor (cell division), if the cells selectedare expected to be actively dividing while in the vehicle, then theywill continue to divide until they fill the available space, or untilphenomena such as contact inhibition limit further division. Forreplicating cells, the geometry and size of the vehicle will be chosenso that complete filling of the vehicle core will not lead todeprivation of critical nutrients due to diffusional limitations. Ingeneral, vehicles that will be filled to confluency with cells or tissuewill be no more than 250 microns in cross-section, such that cells inthe interior will have less than 15 cells between them and an externaldiffusional surface, preferably less than 10 cells and more preferablyless than 5 cells.

In general, for cells not expected to divide within the vehicle, such aschromaffin cells, pancreatic islet cells and the like, the appropriatecell densities will be calculated from the diffusional considerationslisted above.

With respect to the third factor (viscosity of core materials) cells indensities occupying up to 70% of the vehicle volume can be viable, butcell solutions in this concentration range would have considerableviscosity. Introduction of cells in a very viscous solution into thevehicle could be prohibitively difficult. In general, for both two stepand coextrusion strategies, discussed below, cell loading densities ofhigher than 30% will seldom be useful, and in general optimal loadingdensities will be 20% and below. For fragments of tissues, it isimportant, in order to preserve the viability of interior cells, toobserve the same general guidelines as above and tissue fragments shouldnot exceed 250 microns in diameter with the interior cells having lessthan 15, preferably less than 10 cells between them and the nearestdiffusional surface.

Finally, with respect to the fourth factor (preimplantation and assayrequirements), in many cases, a certain amount of time will be requiredbetween vehicle preparation and implantation. For instance, it may beimportant to qualify the vehicle in terms of its biological activity.Thus, in the case of mitotically active cells, preferred loading densitywill also consider the number of cells which must be present in order toperform the qualification assay.

In most cases, prior to implantation in vivo it will be important to usein vitro assays to establish the efficacy of the biologically activemoiety within the vehicle. Vehicles containing the moiety of interestcan be constructed and analyzed using model systems. In a preferredembodiment of the instant invention, the diffusion of glucose into thevehicle is used to stimulate insulin release from pancreatic isletcells. The appearance of insulin outside the vehicle is monitoredthrough the use of an appropriately specific radioimmunoassay. Suchprocedures allow the determination of the efficacy of the vehicle on aper cell or unit volume basis.

Following the above guidelines for vehicle loading and for determinationof vehicle efficacy, the actual vehicle size for implantation will thenbe determined by the amount of biological activity required for theparticular application. In the case of secretory cells releasingtherapeutic substances, standard dosage considerations and criteriaknown to the art will be used to determine the amount of secretorysubstance required. Factors to be considered include; the size andweight of the recipient; the productivity or functional level of thecells; and, where appropriate, the normal productivity or metabolicactivity of the organ or tissue whose function is being replaced oraugmented. It is also important to consider that a fraction of the cellsmay not survive the immunoisolation and implantation procedures, as wellas whether the recipient has a preexisting condition which can interferewith the efficacy of the implant. Vehicles of the instant invention caneasily be manufactured which contain many thousands of cells. Inpreferred embodiments, therapeutically useful immunoisolatory vehiclesused to provide insulin to insulin deficient rats contained on the orderof 1,000 islets. Larger vehicles can also be conveniently prepared bythe method of the current invention.

Because of the potentially large capacity of the immunoisolatoryvehicles, the treatment of many conditions will require only one or atmost a few (less than 10) implanted vehicles to supply an appropriatetherapeutic dose. The use of only a few therapeutically effectiveimplantable vehicles containing a large number of cells provides simpleretrievability which, for many applications, will be preferred overmicrosphere or other small configurations requiring a large number ofvehicles. The immunoisolatory macrocapsule of the present invention iscapable of storing 10,000 to 100,000 cells to as high as 500,000 cellsor more, in individual or cluster form, depending on their type.

This invention also pertains to a method of isolating or protectingbiologically active moieties, such as implanted cells, tissues, or othermaterials from immunological attack. The methods and vehicles of theinstant invention are useful to deliver a wide range of cellularproducts, including high molecular weight products, to an individual inneed of them, and/or to provide needed metabolic functions to anindividual, such as the removal of harmful substances.

Products which can be delivered using the instant vehicle include a widevariety of factors normally secreted by various organs or tissues. Forexample, insulin can be delivered to a diabetic patient, dopamine to apatient suffering from Parkinson's disease, or Factor VIII to a Type Ahemophiliac.

Other products which can be delivered through use of the instant vehicleinclude trophic factors such as erythropoietin, growth hormon, SubstanceP, and neurotensin. This invention is useful for treating individualssuffering from acute and/or chronic pain, by delivery of an analgesic orpain reducing substance to the individual. Such pain reducing substancesinclude enkephalins, catecholamines and other opioid peptides. Suchcompounds may be secreted by, e.g., adrenal chromaffin cells. Anotherfamily of products suited to delivery by the instant vehicle comprisesbiological response modifiers, including lymphokines and cytokines.Antibodies from antibody secreting cells may also be delivered. Specificantibodies which may be useful include those towards tumor specificantigens. The release of antibodies may also be useful in decreasingcirculating levels of compounds such as hormones or growth factors.These products are useful in the treatment of a wide variety ofdiseases, inflammatory conditions or disorders, and cancers.

The instant vehicle can also be used to restore or augment vitalmetabolic functions, such as the removal of toxins or harmfulmetabolites (e.g., cholesterol) from the bloodstream by cells such ashepatocytes. The method and vehicle of the instant invention makepossible the implantation of cells without the concomitant need toimmunosuppress the recipient for the duration of treatment. Through useof the biocompatible immunoisolatory vehicle, homeostasis of particularsubstances can be restored and maintained for extended periods of time.The instant vehicle may contain a multiplicity of cells, such thatimplantation of a single vehicle can be sufficient to provide aneffective amount of the needed substance or function to an individual.

In one embodiment of this invention, methods are provided for theprevention or treatment of neural degeneration. Such neural degenerationoccurs naturally as a result of the aging process, typically inphysically mature individuals, or may occur as a result of aneurological disorder or disease. Examples of human diseases ordisorders which are thought to be associated with neural degenerationinclude Alzheimer's disease, Huntington's chorea, AIDS-related dementia,and Parkinson's disease. These disorders, often occur in physicallymature individuals. However, these and other neurological disorders mayoccur in juveniles.

As used herein, an “aged” individual is an individual in whom neuraldegeneration has occurred or is occurring, either as a result of thenatural aging process, or as a result of a neurodegenerative disorder.Neural degeneration as a result of the natural aging process means lossor decline of neural function compared to a previous state notattributable to a defined clinical abnormality or neurological disorder,such as Alzheimer's, Parkinson's or Huntington's.

Animal models for neurodegenerative conditions are based on the premisethat a specific insult may damage or kill neurons. In some cases thismay even lead to a cascade of neuronal death which affects trophicallyinterdependent neurons along pathways responsible for specific brainfunctions.

A strategy for treatment of neural degenerative condition involves thelocalized administration of growth or trophic factors in order to (1)inhibit further damage to post-synaptic neurons, and (2) improveviability of cells subjected to the insult. Factors known to improveneuronal viability include basic fibroblast growth factor, ciliaryneurotrophic factor, brain-derived neurotrophic factor, neurotrophin-3,neurotensin, and Substance P.

In one animal model for neurodegenerative excitotoxicity, the glutamateanalog, quinolinic acid, is injected stereotaxically into the brainregion known as the striatum and/or basal ganglia to produceneuropathology and symptoms analogous to those of patients sufferingfrom Huntington's disease. Both the model and actual Huntington'sdisease are characterized by damage to neurons necessary for aspects ofmotor control.

Furthermore, one of the early symptoms of Huntington's disease is lossof body weight (Sanberg, P. R. et al. Med J Aust. 1, pp. 407-409 (1981).Similar effects are also seen in the model system (Sanberg, P. R. et al.Exp Neurol, 66, pp. 444-466 (1979). Quinolinic acid is also found atabnormally high levels in AIDS-related dementia.

According to the present invention, trophic factors are provided to theproper brain region by implanting a vehicle containing living cellswhich secret an appropriate factor. Suitably, the cells are adrenalchromaffin cells which are known to secrete at least one factor, basicfibroblast growth factor. Other as yet unidentified trophic factors mayalso be secreted by chromaffin cells. It is to be noted that thisembodiment of the invention is separate from the use of chromaffin cellsto secrete the neurotransmitter, dopamine, for the amelioration ofsymptoms of Parkinson's disease. Nerve growth factor-secreting cellssuch as fibroblasts engineered to express NGF represent an alternativetherapy for this quinolinic acid induced neurodegeneration. Schwanncells prepared from myelin may be encapsulated and implanted inappropriate brain areas to prevent neural degeneration associated withParkinson's disease.

In another embodiment of the invention, the animal model involves lesionof the fimbria-fornix. In particular, neurons of the septohippocampalsystem are axotomized which leads to degeneration and cell death. Thismodel is thought to be analogous to the types of lesions which causeAlzheimer's disease in humans. Suitably, a growth factor, nerve growthfactor (NGF), is provided by the implantation of a vehicle containingcells which secrete NGF. Astrocytes immortalized (e.g. by transformationwith the Large T antigen) and genetically engineered to express NGF maybe employed. Preferably, the cells are fibroblasts which have beengenetically engineered to produce recombinant NGF. The fibroblastssurvive best in a core composed of a matrix material which mimicsextracellular matrix, such as collagen or laminin-containing hydrogels.The core is surrounded by an immunoisolatory jacket which allows thediffusion of oxygen and nutrients to the cells in the core, and alsoallows the secreted NGF to diffuse through the jacket and into the bodyof the recipient. The vehicle implant inhibits the death of cholinergicneurons as assayed by the number of neurons which contain choline acetyltransferase (ChAT), an indicator of viable cholinergic neurons.

Fimbria-fornix lesions also cause behavioral deficits in the animalsubjects of the model, most easily observed in tasks involving learningand memory. It has been reported that chronic administration of NGF torats with fimbria-fornix lesions accelerates the animals' behavioralrecovery (Wills, B. et al. Behav.Brain Res., 17, pp. 17-24 (1985)). Inthe present invention, implantation of the vehicle containingNGF-secreting cells provides a practical way to deliver NGF continuouslyto the appropriate brain region of the lesioned animal. By analogy, thevehicle of the present invention offers a practical form of regenerativeand/or prophylactic therapy for Alzheimer's victims whose conditions maybe ameliorated by continuous delivery of NGF to specific brain regions.

A wide variety of biologically active moieties or cells may be used inthis invention. These include well known, publicly availableimmortalized cell lines as well as primary cell cultures. Examples ofpublicly available cell lines suitable for the practice of thisinvention include, baby hamster kidney (BHK), chinese hamster ovary(CHO), mouse fibroblast (L-M), NIH Swiss mouse embryo (NIH/3T3), Africangreen monkey cell lines (including COS-a, COS-7, BSC-1, BSC-40, BMT-10and Vero), rat adrenal pheochromocytoma (PC12) and rat glial tumor (C6).Primary cells that may be used according to the present inventioninclude, bFGF-responsive neural progenitor-stem cells derived from theCNS of mammals (Richards et al., Proc. Natl. Acad. Sci. USA 89, pp.8591-8595 (1992); Ray et al., Proc. Natl. Acad. Sci. USA, 90, pp.3602-3606 (1993)), primary fibroblasts, Schwann cells, astrocytes, B-TCcells, Hep-G2 cells, AT T20 cells, oligodendrocytes and theirprecursors, myoblasts, adrenal chromaffin cells, and the like.

Schwann cells maybe prepared according to the method of Bunge (PCTpublished application WO 92/03536), mixed with a suitable substratumsuch as Matrigel™, and encapsulated. The encapsulated cells may beimplanted in appropriate areas of the brain to prevent the degenerationof the dopaminergic neurons of the nigral striatal pathway associatedwith Parkinson's disease. Generally, the preferred implant site will bein or near the striatum. Encapsulating the cells may enhance secretionof trophic factors since the cells will not be in proximal contact withneurons, and myelination will not occur. Other glial cell types may beencapsulated and implanted for this purpose, including astrocytes andoligodendrocytes.

The choice of biologically active moiety or cell depends upon theintended application. The encapsulated cells may be chosen for secretionof a neurotransmitter. Such neurotransmitters include dopamine, gammaaminobutyric acid (GABA), serotonin, acetylcholine, noradrenaline,epinephrine, glutamic acid, and other peptide neurotransmitters. Cellscan also be employed which synthesize and secrete agonists, analogs,derivatives or fragments of neurotransmitters which are active,including, for example, cells which secrete bromocriptine, a dopamineagonist, and cells which secrete L-dopa, a dopamine precursor.

The encapsulated cells can also be chosen for their secretion ofhormones, cytokines, growth factors, trophic factors, angiogensisfactors, antibodies, blood coagulation factors, lymphokines, enzymes,and other therapeutic agents or agonists, precursors, active analogs, oractive fragments thereof. These include enkephalins, catecholamines,endorphins, dynorphin, insulin, factor VIII, erythropoietin, SubstanceP, nerve growth factor (NGF), Glial derived Neurotrophic Factor (GNDF),platelet-derived growth factor (PDGF), epidermal growth factor (EGF),brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), anarray of fibroblast growth factors, and ciliary neurotrophic factor.

Alternatively, one or more biologically active molecules may bedelivered into the capsule. For example, the capsule may contain one ormore cells or substances which “scavenge” cholesterol, or otherbiological factors, from the host.

Techniques and procedures for isolating cells or tissues which produce aselected product are known to those skilled in the art, or can beadapted from known procedures with no more than routine experimentation.For example, islets of Langerhans can be isolated from a large-animalpancreas (e.g., human or porcine) using a combination of mechanicaldistention and collagenase digestion, as described by Scharp, D. W., etal., U.S. Pat. No. 4,868,121. Islets may be isolated from small animalssuch as rats by the method of Scharp, et al., Diabetes 29, suppl. 1, pp.19-30 (1980). Similarly, hepatocytes can be isolated from liver tissueusing collagenase digestion followed by tissue fractionation, asdescribed by Sun, A. M., et al., Biomat., Art. Cells, Art. Org., 15, pp.483-496 (1987). Adrenal Chromaffin cells may be isolated by the methodof Livett, B. G., Physiology Reviews, 64, pp. 1103-1161 (1984).

Many cellular products which are difficult to provide using primarydonor tissues can be provided using immortalized cells or cell lines.Immortalized cells are those which are capable of indefinite replicationbut which exhibit contact inhibition upon confluence and are nottumorigenic. An example of an immortalized cell line is the ratpheochromocytoma cell line PC12. Transformed cells or cell lines can beused in a similar manner. Transformed cells are unlike merelyimmortalized cells in that they do not exhibit contact inhibition uponconfluence, and form tumors when implanted into an allogeneic host.Immortalization can allow the use of rare or notoriously fragile cell ortissue types for the long-term delivery of a chosen product or metabolicfunction. Suitable techniques for the immortalization of cells aredescribed in Land H. et al., Nature 304, pp. 596-602 (1983) and Cepko,C. L., Neuron 1, pp. 345-353 (1988). Candidate cell lines includegenetically engineered beta-cell lines which secrete insulin such as NITcells (Hamaguchi, K., et al., Diabetes 40, p. 842 (1991)), RIN cells(Chick, W. L., et al., Proc. Natl. Acad. Sci. USA, 74, pp. 628-632(1977)), ATT cells (Hughes, S. D., et al, Proc. Natl. Acad. Sci. USA,89, pp. 688-692 (1992)), CHO cells (Matsumoto, M., et al, 1990, Proc.Natl. Acad. Sci. USA, 87, pp. 9133-9137 (1990)), and beta-TC-3 cells(Tal, M., et al, 1992, Mol. Cell Biol., 12, pp. 422-432 (1992)).Additionally, recombinant cells or cell lines can be engineered toprovide novel products or functions and combinations thereof, using awide variety of techniques well known to those of ordinary skill in theart.

For example, fibroblasts can be transfected with an expression vectorfor the chosen product (e.g., nerve growth factor, erythropoietin,insulin, or Factor VIII). It should be recognized however, thatexpression of a recombinant protein in a cell type which does notnormally express the protein may lead to unregulated expression whichmay not be desirable for certain medical applications.

B-cell hybridomas secreting a selected monoclonal antibody, or T-cellhybridomas secreting a selected lymphokine, can also be used. It may beparticularly desirable to deliver a monoclonal antibody or fractionthereof, which neutralizes the biological activity of a disregulatedbiological response modifier using the instant invention. Engineeredcells which secrete soluble fragments of receptors for these biologicalresponse modifiers can be used in a similar fashion. The disregulationor overproduction of particular biological response modifiers has beenimplicated in the etiology of certain cancers.

The encapsulated material can be tissue or cells able to secrete suchantinociceptive agents, including any one of catecholamines,enkephalins, opioid peptides or mixtures thereof. Preferablycatecholamines are secreted, most preferably a mixture of catecholaminesand enkephalins. Typically, the encapsulated material can be tissue ofthe adrenal medulla, or more particularly, adrenal medulla chromaffincells. Additionally, genetically engineered cell lines or othernaturally occurring cell lines able to secrete at least one painreducing agent such as a catecholamine, enkephalin, opioid peptide, oragonist analog thereof, can be used.

If the cells to be immunoisolated are replicating cells or cell linesadapted to growth in vitro, it is particularly advantageous to generatea cell bank of these cells. A particular advantage of a cell bank isthat it is a source of cells prepared from the same culture or batch ofcells. That is, all cells originated from the same source of cells andhave been exposed to the same conditions and stresses. Therefore, thevials can be treated as identical clones. In the transplantationcontext, this greatly facilitates the production of identical orreplacement immunoisolatory vehicles. It also allows simplified testingprotocols which assure that implanted cells are free of retroviruses andthe like. It may also allow for parallel monitoring of vehicles in vivoand in vitro, thus allowing investigation of effects or factors uniqueto residence in vivo.

In all cases, it is important that the cells or tissue contained in thevehicle are not contaminated or adulterated. If a vehicle having amatrix core is desired, the cells are mixed under sterile conditions,with an appropriate amount of a biocompatible, gellable hydrogel matrixprecursor. There are numerous natural and synthetic hydrogels which aresuitable for use in a biocompatible immunoisolatory vehicle of theinstant invention. Suitable naturally-derived hydrogels includeplant-derived gums, such as the alkali metal alginates and agarose, andother plant-derived substances, such as cellulose and its derivatives(e.g., methylcellulose). Animal tissue-derived hydrogels such as gelatinare also useful. Alternatively, the core matrix can be made ofextracellular matrix (ECM) components, as described by Kleinman et al.,U.S. Pat. No. 4,829,000. Suitable synthetic hydrogels include polyvinylalcohol, block copolymer of ethylene-vinylalcohol, sodium polystyrenesulfonate, vinyl-methyl-tribenzyl ammonium chloride and polyphosphazene(Cohen, S. et al. J. Anal. Chem. Soc., 112, pp. 7832-7833 (1990)).

The newly-formed immunoisolatory vehicle obtained by any of the methodsdescribed herein can be maintained under sterile conditions in anon-pyrogenic, serum-free defined nutrient medium or balanced saltsolution, at about 37° C., prior to implantation. Lower temperatures(20° C.-37° C.) may be optimal for certain cell types and/or culturingconditions. Other holding temperatures and medium compositionsconsistent with good cell viability may also be used. Alternatively, thevehicle can be cryopreserved in liquid nitrogen, if a cryoprotectiveagent such as glycerin has been incorporated into the matrix (Rajotte,R. V. et al. Transplantation Proceedings, 21, pp. 2638-2640 (1989)). Insuch a case, the vehicle is thawed before use and equilibrated understerile conditions as described above.

Implantation of the biocompatible immunoisolatory vehicle is alsoperformed under sterile conditions. Generally, the immunoisolatoryvehicle is implanted at a site in the recipient's body which will allowappropriate delivery of the secreted product or function to therecipient and of nutrients to the implanted cells or tissue, and willalso allow access to the vehicle for retrieval and/or replacement. It isconsidered preferable to verify that the cells immobilized within thevehicle function properly both before and after implantation; assays ordiagnostic tests well known in the art can be used for these purposes.For example, an ELISA (enzyme-linked immunosorbent assay),chromatographic or enzymatic assay, or bioassay specific for thesecreted product can be used. If desired, secretory function of animplant can be monitored over time by collecting appropriate samples(e.g., serum) from the recipient and assaying them.

The invention will now be further illustrated by the following examples,which are not to be viewed as limiting in any way.

EXAMPLE 1 Preparation of Cells For Immunoisolation

Cells derived from cell lines or from primary sources were maintained invitro prior to immunoisolation. (In some cases, cells can be storedcryopreserved and then thawed and acclimated in vitro.) Conditions forincubation will vary for specific cell types, but will be readilyascertainable to those skilled in the art following no more than routineexperimentation. Islets of Langerhans were obtained by the methods ofScharp et al.(supra) and maintained at 37° C. in an atmosphere of 5%CO₂-95% air in a medium consisting of a nutrient broth (e.g., Ham's F12(GIBCO)), supplemented with serum (e.g., 25% v/v pooled equine donorserum). Islets were maintained in culture, using Petri dishes at 24° C.for predetermined periods of time, according to the method of Lacy, P.E. et. al., Science, 204, pp. 312-313 (1979). Prior to immunoisolation,the islets were collected, concentrated by swirling the Petri dish,resuspended in Hank's balanced salt solution (HBSS). The washed isletswere resuspended in a sufficient volume of HBSS to yield the final isletdensity required to form an immunoisolatory vehicle containing thedesired number of islets, of an appropriate size and shape forimplantation and subsequent restoration of normoglycemia to a diabeticindividual. This method of preparing the cells prior to immunbisolationis thought to remove antigen presenting cells from the islet tissue thusdiminishing immunologic attraction to the outside of the vehicle whichcould limit its function and duration.

EXAMPLE 2 Formation of Hydrogel Matrices with Different Molecular WeightCutoffs

An alginate thin film made from a solution of 1.0% w/v sodium alginatein H₂O was cross-linked for 6 minutes using either (1) a 1.0% (w/v) or(2) a 2.0% (w/v) aqueous CaCl₂ solution. The sheet was made by placing afilm of liquid on a glass plate using a draw down blade with a 0.2 mmclearance, then immersing in the aqueous CaCl₂ solution. A disk was cutfrom the film using a 47 mm cutting die. The disk was placed in anAmicon stirred filtration cell and used to filter solutions of severalmarker solutes under a pressure of 10 psi. The concentration of themarker solute was measured in the retentate (C_(r)=average of initialand final retentate concentration) and similarly in the bulked permeate(C_(p)). The rejection coefficient of each hydrogel was calculated asfollows:

 R=1−C _(p) /C _(r)

Thus, a solute which is completely rejected would have a coefficient of1, and conversely, one which is completely passed through the hydrogelwould have a coefficient of 0. The hydrogel resulting from (1) waspermeable to 2,000 kD Dextran (Poly Sciences corp.) (rejectioncoefficient equal to 0.64). The hydrogel resulting from (2) was nearlyimpermeable to the same Dextran solution. FIG. 1 describes permeabilityof the two hydrogels to the following additional solutes. Bovine SerumAlbumin (BSA; ICN Biochemicals), Vitamin B12 (ICN Biochemicals),α-chymotrypsin (ICN Biochemicals), Apoferritin (Sigma). The approximatemolecular weights are given in parenthesis in FIG. 1.

EXAMPLE 3 Formation of a Dual-Matrix Immunoisolatory Vehicle

A 2% solution of sodium alginate in physiological saline (PS; 150 mmNaCl) was prepared under sterile conditions. A sterile suspension ofpancreatic islets in CRML1066 (GIBCO) culture media isolated from adultrats was diluted 1:1 with the alginate solution, for a finalconcentration of 1% alginate in the islet suspension. The isletsuspension was extruded from a single chamber extrusion nozzle into a 1%CaCl₂ bath. Once the alginate polyions were crosslinked (approx. 2 min.)and the core containing immobilized islets formed, the core was placedin a 2% alginate solution. The core was then drawn up into tubing with adiameter approximately 500 microns larger then the core and reextrudedinto a second crosslinking bath of 2% alginate, surrounding the corewith a jacket formed of a separate cell-free layer of alginate matrixcross-linked to the core. The thus formed macrocapsule was cylindricalwith dimensions of 30 mm length, 800 mm core diameter, and 1 mm diametercore to jacket. The core volume was 15 mm. The core contained 300 isletsconstituting a volume fraction of 10.6% of the total core volume.

EXAMPLE 4 Formation of a Dual-Matrix Immunoisolatory Vehicle byCoextrusion

A 2% solution of sodium alginate in physiological saline (PS; 150 mmNaCl) was prepared under sterile conditions. A sterile suspension ofpancreatic islets in CRML1066 culture media isolated from adult rats wasdiluted 1:1 with the alginate solution, for a final concentration of 1%alginate in the islet suspension.

The islet suspension was loaded into the inner chamber of a nesteddual-bore coextrusion device of the configuration described previously,the inner bore of which has a diameter of 500 microns and the peripheralbore of which has a diameter of 600 microns. The outer chamber of thisdevice was loaded with a solution of sterile 1% sodium alginate in PS.

The tip of the nozzle was immersed in a bath containing a sterilesolution of 1% CaCl₂ in PS, which induces the hardening or gelling ofalginate by crosslinkage of alginate polyions. The materials loaded intothe chambers were coextruded into this bath, generating a continuouslyforming alginate cylinder containing a core region of alginatematrix-immobilized islets and a surrounding region of alginate matrixfree of islets. The outer diameter of the jacket was 1.2 mm. The innerdiameter of the core was 1.0-1.05 mm. The total islet volume of the corewas 0.8 mm³ (200 islets). The total core volume was 25.98 mm³. Thevolume of the islets was 3% of the total core volume. The MWCO for 1%alginate was as depicted in FIG. 1. However, th MWCO will increase withtime similarly to FIG. 7 due to continued Ca⁺⁺ displacement. Thealginate of the core was cross-linked with the alginate of the jacket.

The relative thickness of the surrounding region was modified byadjusting the speeds at which the materials were extruded from the coreand peripheral bores of the nozzle. In general as flow in the coreincreased, wall thickness decreased. As flow in the peripheral bore wasincreased wall thickness increased, Ranges of flow rates used were thesame for both the core and periphery (0.3-1.5 ml/min.). The ends of thecylinder were sealed by dipping the cylinder first in a sterile 2%sodium alginate bath, then in a sterile 1% CaCl₂ bath. Theimmunoisolatory vehicle so formed was maintained in sterile tissueculture medium prior to implantation.

EXAMPLE 5 Formation of Core Matrix, Permselective MembraneImmunoisolatory Vehicle by Coextrusion

A suspension of rat islets in 1% alginate was prepared as described inExample 3 and loaded into the inner chamber of the coextrusion device.The outer chamber was loaded with a membrane casting solution comprising12.5% (w/w) PAN/PVC in DMSO (i.e., dimethyl sulfoxide). The tip of thenozzle was either positioned at a fixed distance above, or immersed in,a bath containing a sterile solution of 1% CaCl₂ in PS, which inducedthe hardening or gelling of the alginate core matrix, and simultaneouslyinduced the hardening of the casting solution into a permselectivemembrane. The exterior surface characteristics of the vehicle weredetermined by whether the nozzle had been positioned above or below thesurface of the bath. When the nozzle was positioned above the bath andexposed to low relative humidity (RH) air, an anisotropic membrane witha rough, coriaceous (i.e., leathery) external surface was formed, butwhen the nozzle was immersed in the bath, a membrane-bilayer with asmooth external surface was formed. Alternatively, when the nozzle wasplaced above the bath and the fiber exposed to high RH air, anintermediate was formed. The materials loaded into the chambers werecoextruded into this bath, generating a continuously forming cylindricalvehicle, comprising a core region of alginate matrix-immobilized isletsand a surrounding semipermeable membrane having a MWCO of 50 kD.

The relative thickness of the membrane was modified by adjusting therelative velocity of extrusion from the bores, as described in Example3. The ends of the cylinder were sealed using methods similar to thosedescribed in the copending U.S. patent application Ser. No. 07/461,999filed Jan. 8, 1990, the teachings of which are herein incorporated byreference. The immunoisolatory vehicle so formed was maintained insterile PS, a balanced salt solution or tissue culture medium prior toimplantation.

EXAMPLE 6 Formation of Core Matrix, Permselective MembraneImmunoisolatory Vehicle by “Hand Loading”

In other cases, the islets were suspended in a 1-2% alginate solution,and “hand loaded” into preformed thermoplastic hollow fibers using asyringe and the ends of the fibers were sealed by a combination of heatand polymer glue precipitation as described in copending U.S. Ser. No.07/461,999. The MWCO of the thermoplastic jacket was approximately 50kD. The hydrogel matrix was formed by incubating the loaded fibers in a1% calcium chloride solution for 6 minutes.

EXAMPLE 7 Assessment of Viability and Function of Immobilized,Immunoisolated Islets In Vitro

Adult rat islets immunoisolated within double matrix vehicles by themethods described in Examples 3 and 6. Matrix liquid core vehicles withthermoplastic jackets were incubated in vitro for at least two weeks.The vehicle had a core outer diameter of 800 μm overlain with a wallthickness of 65 μm. Alginate/Alginate dual matrix vehicles had an 880 μmcore diameter and 60 μm wall thickness. Incubation conditions were:immersion in Ham's F12 medium supplemented with 25% equine serum at 37°C. in a 5% CO₂ -95% air atmosphere. The medium was refreshed every threeto four days.

Using propidium iodide, the immunoisolated islet cells were found to be95% viable after this incubation period in vitro. They were shown toremain functional as well. When tested by perfusion with glucose(Dionne, supra) immunoisolated islets were shown to have an insulinsecretory response similar both in magnitude and pattern to that ofunprotected islets incubated in vitro for a similar period of time andunder similar conditions. Insulin release was measured by the method ofSoeldner, J. S. et al. Diabetes, 14, p. 771 (1965). The results of atypical perfusion experiment are shown in FIGS. 2A and 2B. The challengeand baseline concentrations of glucose used were 300 mg % and 100 mg %,respectively. No significant delay in either the onset of the firstphase of insulin release following glucose stimulation or return tobaseline secretion was observed with immunoisolated islets. In addition,a rising second phase comparable to that of unprotected islets was seen.Expressed on a per-islet basis, the total amounts of insulin released byimmunoisolated islets were similar to that for unprotected islets. Theseresults are summarized in FIGS. 3A and 3B.

EXAMPLE 8 Comparison of In Vivo Performance of Xenografted IsletsIsolated Within Vehicles Having a Thermoplastic Membrane With andWithout an Internal Hydrogel Matrix

Rat islets were immunoisolated in either matrix or liquid corethermoplastic vehicles as described in Example 6. Vehicle dimensionswere 800 μm O.D., 55 μm wall thickness, 2 cm fiber length. Just under20% loading density was used. In the case of liquid corecapsules,alginate was not included in the cell suspension. In the firstexperiment, islets were immunoisolated within a matrix. Vehicles wereimplanted intraperitoneally into streptozotocin-induced diabetic micefor a concordant xenograft (i.e., between closely-related species).Free-floating implants were inserted into the peritoneal cavity. Eightanimals were implanted with 500-1000 immunoisolated rat islets each. Oneanimal showed no amelioration of hyperglycemia. The others returned to anormoglycemic state (i.e., the plasma glucose levels of these animalsreturned to a normal range defined as 100-125 mg %) within five daysposttransplantation and remained normoglycemic until day 60 when thegrafts were removed and the animals again became hyperglycemic. Theaverage results of 7 such experiments are summarized in FIG. 4. Theabsence of significant fluctuations in plasma glucose levels in theseanimals should be noted. The recovered immunoisolatory vehicles wereinspected for evidence of fibrotic overgrowth, and were assessed for theability to release insulin in response to glucose perfusion. None of thevehicles had become completely encapsulated with fibroblasts, however insome areas three to five layers of fibroblasts around the exterior ofthe vehicle were observed. Recovered immunoisolatory vehicles releasedinsulin in vitro following perfusion in response to glucose andtheophylline stimulation and histological analysis revealed viableislets with evidence of insulin staining within the cells. The resultsof the perfusion experiment with glucose and theophylline stimulationare shown in FIG. 5.

These favorable results contrasted sharply with those obtained when ratislets were immunoisolated within a PAN/PVC membrane without animmobilizing matrix. For these immunoisolatory vehicles, functionalresponsiveness lasted only 12±3 days post implantation; five out of fiveanimals tested returned to a hyperglycemic state thereafter.Histological examination of these immunoisolatory vehicles revealedagglomeration of the islets. The islets had condensed into a large massof tissue which exhibited severe central necrosis, with only a rim ofviable-and identifiable islet cells surviving. Thus, the presence of amatrix to prevent islet aggregation and resulting cell death,significantly improved viability resulting in long term efficacy of theimplant.

EXAMPLE 9 Assessment of the Restoration of Normoglycemia toStreptozotocin-Induced Diabetic Mice by Implantation of ImmunoisolatedConcordant Xenoaraft Islets in Immunoglobulin Permeable Vehicles

In vivo performance of the double-matrix immunoisolatory vehicle wasassessed using the rat-to-mouse concordant xenograft model forrestoration of normoglycemia to streptozotocin induced diabetic mice.The vehicles were prepared as in Example 3 and have significantpermeability to 2,000 kD Dextran (FIG. 1). Therefore, these vehicleswere also readily permeable to IgG (150 kD). The results of thisexperiment are summarized in Table 2. The animals were divided intothree groups: group 1 consisted of seven control animals into whom 300nonimmunoisolated islets per animal were implanted at the kidneysubcapsular site. Only one of these animals showed amelioration ofhyperglycemia for more than 12 days. The mean duration of normoglycemiain Group 1 was 14.0±3.1 days.

Group 2 consisted of thirteen animals each implanted with 300 rat isletsimmobilized in an alginate matrix lacking a surrounding cell-freeregion. Graft function was lost within 24 days in 8/13 of these Group 2animals, indicating that simple immobilization conferred no advantageover the Group 1 controls. The other five animals remained normoglycemicuntil the graft was removed. The mean duration of normoglycemia in group2 was 29.3±5.5 days.

Group 3 consisted of ten animals, each implanted with 300 rat isletsimmobilized within a dual-matrix immunoisolatory vehicle of theconfiguration described in Example 3, i.e., with a surrounding layer ofcell-free alginate matrix. In only four of these animals, graft functionwas lost within 14 days postimplantation. However, six animals remainednormoglycemic beyond 100 days at which time the experiment wasterminated and the grafts removed, precipitating a return to thediabetic state (FIG. 6). The mean duration of normoglycemia in group 3was 65.8±15.1 days (n=10). Fibromatous reaction to the recoveredalginate vehicle was minimal. Histological analysis of recoveredimmunoisolated islets revealed viable islets with evidence of insulinstaining within the β cells. The immunoisolatory vehicles used in thisexperiment also demonstrate the functionality and biocompatibility ofvehicles which are permeable to high molecular weight proteins such asthe immunoglobulin G protein.

TABLE 1 SURVIVAL OF IMMUNOISOLATED XENOGRAFTS IN DIABETIC MICE Survival(Days) Group Vehicle Individual Group 1 control 7, 12, 12, 12,  14.0 ±3.1 12, 12, 29 2 immobilized 8, 12, 12, 14, >29.3 ± 5.5 15, 16, 18, 24,41*-, 53*, 54*, 54*-, 60*- 3 immuno- 8, 8, 12, 13, >65.8 ± 15.1 isolated102*-, 102*-, 102*-, 102*-, 104*-, 104*- *Nephrectomy for removal ofislet graft

A vehicle of the configuration described in Example 3 was prepared; itcontained several hundred islets and had a membrane MWCO of less than 50kD.

The vehicle was implanted into a diabetic BB rat. This strain of rat isknown to be a rodent model for mimicking human Type 1 autoimmunediabetes. The vehicle was recovered after a 21-day period of residencein vivo. The immunoisolated islets were found to be viable andfunctional, as determined by histological analysis.

EXAMPLE 10 Assessment of the Survival of Immunoisolated Islets in aDiscordant Xenogenic Recipient

Dual matrix immunoisolatory vehicles containing immobilized rat isletswere prepared by the process of coextrusion from a nested dual-borenozzle as described in Example 4. The conditions for the gelling of thematrix were chosen to yield a hydrogel matrix permeable to 2,000 kD blueDextran (as in FIG. 7), thus the vehicle so formed was permeable toimmunoglobulin G and to C1q.

Segments of about 0.5 cm in length were prepared from the continuouscylindrical vehicle by periodically interrupting the flow of the corematerial to form cell free regions, which were readily visible. Thefiber was cut in the cell free region; thus the cells were completelysurrounded by a region of cell-free alginate matrix. The vehicles wereimplanted between the leaves of the mesentery of guinea pigs (n=2), adiscordant (i.e., distantly related) host. After 21 days of residence invivo, the vehicles were removed and tested in vitro forglucose-responsive insulin release. These results are summarized in FIG.8. Following basal stimulation, a statistically significant rise ininsulin release from the immunoisolated islets was measured whenstimulated with 300 mg/dl glucose. A return to basal insulin levelsoccurred when glucose returned to 100 mg/dl. Thermoplastic vehicles withalginate cores gave similar results.

EXAMPLE 11 Improved Tissue Viability by Controlled Reaggregation

Purified canine islets, prepared according to the method of Scharp andLacy, U.S. Pat. No. 5,079,160, were dispersed into cell aggregatescontaining from one to 50 cells according to the following protocol.1000 canine islets were rinsed 3 times with 50 ml of Ca++ and Mg++ freeHanks medium containing 1 mm EDTA. After the final rinse, islets werecentrifuged into a pellet at 100×g for 8 min., 10° C. The resultingpellet was resuspended in 5 ml of the same medium per 1000 islets. Thisslurry was agitated for 8 min. at 24° C. using a hand held micro-pipet.At the end of 8 min., trypsin and DNAse were added to finalconcentrations of 25 ug/ml and 2 ug/ml respectively. The slurry wasfurther agitated by repeated pipetting for approximately 5 min at whichtime microscopic examination indicated that the largest aggregatesconsisted of no more than about 50 cells. The digestion was quenched byadding 10 ml of cold DMEM with 10% new born calf serum per 1000 islets.Aggregates were pelleted by centrifugation at 250×g for 6 min.Aggregates were cultured in Ham's F12 with 25% horse serum overnightduring which time limited reaggregation occurred resulting in a volumeaverage aggregate size of approximately 35 um.

Following overnight culture, aggregates were pelleted by centrifugationat 250×g for 6 min. A 2% solution of non-crosslinked alginate was addedto the pellet to make a solution consisting of 1% alginate and 8% tissue(w/v). The final islet volume of the core was 0.56 mm³ in the form ofaggregates evenly dispersed throughout the viscous liquid. The tissueslurry was aseptically aspirated into a length of Pe 90 tubing the endof which was previously necked down so as to fit into the lumen of 670μm inner diameter hollow fiber membranes (outer diameter=800 μm. Tenmicroliters of tissue containing slurry was injected into each ofseveral PAN/PVC fibers of 2 cm length. The ends of each fiber weresealed as described previously and the fibers were stored overnight inHam's F12 with 25% horse serum. Prior to implantation, fibers wereplaced into fresh Hanks without serum for 1 hour in order to removeserum residue.

After overnight culture, ½ of the vehicles were implanted into theperitoneal cavity of normoglycemic rats (2 vehicles per animal) and ½ ofth vehicles were maintained in in vitro tissue culture. After 2 weeks,implanted vehicles were removed from the peritoneal cavity and subjectedto an in vitro glucose challenge along with control vehicles cultured invitro. Explanted vehicles exhibited insulin release that was as good asor better than that seen prior to implantation indicating functionalsurvival not only of insulin release but of glucose sensitivity.Following glucose challenge, the alginate core of the vehicles was“blown out” and the tissue was stained with PI for viability. Althougheach aggregate had pulled together to form a tight spheroid ofapproximately 35 um in diameter, individual aggregates remained welldistributed throughout the alginate core and had not clustered togetherto form necrotic regions. Failure to stain with PI stain indicated 100%viability.

EXAMPLE 12 Partial Restoration of Motor Behavior Upon Implantation ofImmunoisolated Adrenal Chromaffin Cells in an Experimental Model ofParkinsonism

Bovine adrenal chromaffin cells were recovered from adrenal glandsaccording to the method of Livett B. G. Physiol. Rev., 64, pp. 1103-1161(1984) by collagenase digestion and maintained in culture for 10 days toensure lack of bacterial or fungal contamination. Clumps of chromaffincells were washed in serum-free medium by centrifugation and resuspendedin 1% alginate solution. This cell suspension was used to form amatrix-core, thermo-plastic membrane immunoisolatory vehicle byco-extrusion as described in Example 4. The aqueous precipitation bathcomprised a solution of 1% CaCl₂ mixed 1:2 with tissue culture mediumfor a final concentration of 0.5% CaCl₂ in the bath. The fiber wasincubated in the bath for 6 minutes to allow the alginate to gel, andwas then transferred to a petri dish containing Dulbecco's modifiedEagle's medium (DMEM). The fiber was visually inspected for regions withgood wall morphology, then spot-checked for the presence of chromaffincells. It was then divided into 4 mm long sections by sealing the endsof the sections with a combination of heat, solvent, and pressure.

Eight Sprague-Dawley rats received injections (10 μg/5 ul) of6-hydroxydopamine (6-OHDA) into the substantia nigra. They were testedfor apomorphine-induced (0.1 mg/kg) rotational behavior at weeklyintervals. By the method of Ungerstedt V. U., Acton. Physiol. Scand.Suppl., 367, pp. 69-93 (1971) and Ungerstedt V. U., Brain Res., 24, pp.485-493 (1970). Apomorphine induces the Parkinson's-like motor responseof turning away from the side of the 6-OHDA-induced lesion. The extentof such rotational motor behavior upon apomorphine injection can be usedto monitor the extent of the lesion and the degree of ameliorationprovided by the immunoisolated chromaffin cells. Six weeks after theinduction of Parkinsonism, the animals received intrastriatal implantsof either control (empty) or adrenal-chromaffin cell-containingvehicles, and were again tested for rotational behavior at weeklyintervals. These results are summarized in FIG. 9.

Prior to implantation, all animals exhibited an equivalent number ofrotations following apomorphine challenge. Within two weekspostimplantation, however, those animals receiving immunoisolatedadrenal chromaffin cells exhibited a significant 35-40% percent decreasein rotations which remained stable throughout testing; this indicatesthat the implants significantly reversed the effects of 6-OHDA-inducedlesions. The animals who received control vehicles did not show anyreduction in the number of rotations.

EXAMPLE 13 Implantation of Adrenal Chromaffin Cells for Prevention ofNeural Damage Due to Excitotoxicity (Huntington's Disease Model)

This example sets forth a method for prevention of neural damage due toneural excitotoxicity in a subject by implantation of a vehiclecontaining cells which secrete a trophic factor. This animal model ofneuroexcitotoxicity is considered analogous to the types of neuraldamage suffered by patients with Huntington's disease.

Subjects

Male Sprague-Dawley rats (N=23) 90-120 days old and weighingapproximately 225-250 grams were used in the following experiments. Allanimals were housed groups of 2-3 in a temperature and humiditycontrolled colony room maintained on a 12 hour light/dark cycle withlights on at 0700 hours. Food and water were freely available duringtesting.

Preparation of Adrenal Cell-containing Macrocapsules

Bovine adrenal chromaffin cells were isolated from adrenal glands andmaintained in culture for 10 days to ensure lack of bacterial or fungalcontamination. Clumps of chromaffin cells were washed in serum-freemedium by centrifugation and resuspended in 1% alginate solution. Thiscell suspension was used as the bore solution for coextrusion in 15%PAN/PVC:DMSO solution. The coextruded fiber containing chromaffin cellswas collected in a bath of 1% CaCl₂ mixed 1:2 with tissue culturemedium. The fiber was left in that solution for 6 minutes to allow thealginate to gel, and was then transferred to a petri dish containingmedium. Fiber was visually inspected for regions with good wallmorphology, then spot-checked for the presence of chromaffin cells.Capsules were made by sealing the ends of 4 mm long sections with acombination of heat, solvent, and pressure. Capsules were then implantedstereotaxically into the brains of Sprague Dawley rats.

Preparation of Quinolinic Acid

Quinolinic acid (Sigma Chemical Co) was dissolved in 2N sodium hydroxideand diluted with phosphate buffer at pH 7.2 to a final pH of 7.4 andconcentration of 225 nmole/1 ul.

Surgical Procedure

Rats were anesthetized with sodium pentobarbital (45 mg/kg) andpositioned in a Kopf stereotaxic apparatus. A sagittal incision was madein the scalp and a hole drilled in the skull for placement of themacroencapsulated adrenal chromaffin cells. Capsules were placed in acapillary tube mounted to the stereotaxic device and lowered to thefollowing coordinates: 0.3 mm anterior to Bregma, 3.5 mm lateral to thesagittal suture and 7.5 mm ventral from the surface of the brain.

One week later, the animals were anesthetized and mounted in thestereotaxic instrument prior to intrastriatal administration ofquinolinic acid or the phosphate buffer vehicle. Solutions were slowlyinfused (0.125 ul/minute) using a 10 ul Hamilton syringe) through a holedrilled 0.8 mm lateral to the site of placement of the capsule. Theinjection site for quinolinic acid was: 0.3 mm anterior to Bregma, 2.7mm lateral to the sagittal suture, and 5.5 mm ventral from the surfaceof the brain. A total volume of 1.0 ul was delivered and the injectioncannula was left in place for an additional 2 minutes to promote thelocal diffusion of the perfusate. This procedure resulted in theformation of 3 experimental groups: adrenal capsule/quinolinic acid(N=8), empty capsule/quinolinic acid (N=8), quinolinic acid only (N=7).

Body weights were recorded on a daily basis for 15 days followingquinolinic acid administration.

Histology

Thirty days following quinolinic acid administration, animals weretranscardially perfused using a peristaltic pump, with 0.9% saline (50ml/min) followed by 1% paraformaldehyde/1.25% glutaraldehyde in 0.1 Mphosphate buffer (4° C.) (800 ml/30 minutes). The brains were thenpostfixed for 2 hours in the paraformaldehyde solution prior to beingplaced in 20% sucrose for 24 hours. The brains were then frozen andserially cut on a sliding microtome into 30 um coronal sections.Sections were then processed for cytochrome oxidase histochemistry andadjacent sections were stained for Nissl.

Results

The presence of cytochrome oxidase was considered indicative ofmetabolic activity, and thus of neuronal viability. Nissl staining wasused to visualize the cell processes and to assess the general structureof the neural architecture.

In the experimental group that received empty capsules, the striatum wasshrunken 20-40% as compared with the lesioned animals that had receivedvehicles containing chromaffin cells. The striatal neurons of the emptycapsule group showed lack of metabolic activity as demonstrated by theabsence of staining for cytochrome oxidase. Furthermore, all animalsshowed significant decrease in body weight (FIG. 10).

In contrast, the neurons of the group which received vehicles containingchromaffin cells showed normal staining with both cytochrome oxidase andNissl, and no loss of body weight.

Conclusion

The neurons of subjects which received chromaffin cell-containingimplants were protected from excitotoxic damage caused by quinolinicacid.

EXAMPLE 14 Treatment of Neural Degeneration in Rats Using EncapsulatedGenetically Engineered Fibroblasts

This example sets forth a method of treatment for animals with afimbria-fornix lesion. This type of lesion produces neuron cell deathand degeneration of post-synaptic neurons and behavioral symptomsindicating deficits in memory and learning. The degeneration ofcholinergic neurons produced in this animal model are consideredanalogous to similar effects seen in Alzheimer's disease in humans.

Surgical Procedure

Adult male Sprague-Dawley rats (250-350 g) were anesthetized byintraperitoneal injection of sodium pentobarbital (55 mg/kg). Unilateralfimbria-fornix lesions were performed by aspiration of the fimbria,dorsal fornix, medial part of the parietal cortex, ventral hippocampalcommissure, corpus callosum, and overlying cingulate cortex. An implantvehicle as described below was then placed into the ipsilateral lateralventricle of each animal, oriented perpendicular to the interauralplane.

Preparation of PAN/PVC Fibers

Permselective hollow fibers were prepared via the dry jet-wet spinningtechnique. Cabasso, Hollow Fiber Membranes, vol. 12, Kirk-OthmerEncyclopedia of Chemical Technology, Wiley, N.Y., 3rd Ed., pp. 492-517(1980). A 15% solution of PAN/PVC copolymer in dimethylsulfoxide (DMSO)was extruded through an annular spinneret, with solvent DMSO (for aporous inner surface) or nonsolvent water (for a smooth skinned innersurface) flowing through the bore. The resulting fiber was collectedinto a nonsolvent water bath, glycerinated, and dried.

Genetically Engineered Fibroblasts

R208N.8 and R208F rat fibroblasts were a gift of Dr. Xandra Breakefield,Harvard University. R208N.8 fibroblasts were engineered to secrete NGFas follows (Short et al., Dev. Neurosci., 12, pp. 34-45 (1990)). Aretroviral vector was constructed from Moloney murine leukemia virus. Itcontained the last 777-basepairs of the coding region for mouse NGF cDNAunder control of the viral 5′ long terminal repeat. The vector alsoincluded a dominant selectable marker encoding the neomycin-resistancefunction of transposon Tn5 under control of an internal Rous sarcomavirus promoter. Transmissible retrovirus was produced by transfectingvector DNA-into PA317 amphotropic producer mouse fibroblast cells and byusing medium from these cells to infect ¥2 ecotropic cells. Virus fromthe ¥2 clone producing the highest titer was used to infect theestablished rat fibroblast cell line R208F, transforming it to R208N.8.Individual neomycin-resistant colonies, selected in medium containingthe neomycin analog G418, were expanded and tested for NGF productionand secretion by a two-site enzyme immunoassay.

Encapsulation of Fibroblasts

R208F and R208N.8 fibroblasts were dissociated with Trypsin-EDTA, andsuspended at a density of 1×10⁶ cells/μl in a laminin containingmatrigel resuspended in Matrigel™ or Vitrogen™, and aspirated with a 1cc syringe into a pre-sterilized PAN/PVC hollow fiber. Fibers were cutto 4 mm length, and the fiber ends were heat sealed with a sterilesurgical cautery.

ChAT Immunocytochemistry

After two weeks, the animals were sacrificed, fixed by transcardialperfusion with cold heparinized physiologic saline and 4%paraformaldehyde in phosphate buffer. The brains were immediatelydissected and postfixed overnight, followed by immersion in 15% and 25%buffered sucrose solutions. Frozen sections were cut at 25 μm fromanterior to posterior on a cryostat, and all coronal sections werecollected onto slides or into phosphate buffer. Representative coronalsections were processed for immunocytochemistry using a monoclonalantibody to rat ChAT (2.5 μg/ml) with the biotin-avidin-DAB method.Sections were mounted and neuronal cell bodies counterstained withcresyl violet. All ChAT-positive cell bodies were counted in the medialseptum and vertical diagonal band region ipsilateral and contralateralto the lesion, between the genu of the corpus callosum and thedecussation of the anterior commissure. A significant prevention ofChAT(+) cell reduction was observed in rats receiving R208N.8 capsules.

EXAMPLE 15 Use of an Immunoisolatory Vehicle to Deliver a High-MolecularWeight Product to a Recipient

Immunoisolatory vehicles were prepared by hand loading 350,000 hybridomacells producing an antibody (iostype immunoglobulin G), specific fortumor necrosis factor (TNF) into a 7 mm length of a medical grade,olefinic microporous hollow fiber of the kind used for plasmapheresis(Plasmaphan; Enka). The internal diameter of the fiber was 300 microns;its MWCO was about 1,000 kD. The ends of the vehicle were sealed asdescribed in copending U.S. Ser. No. 07/461,999. The vehicle wasimplanted under the renal capsule of a mouse, where it was allowed toreside for 14 days. The vehicle was thereafter recovered and found tocontain many cells, over 50% of which were viable as determined by theexclusion of indicator dye (pI). The release of TNF-specific antibodyinto the serum of the recipient mouse was monitored by ELISA. Theresults are summarized below:

TABLE 2 Days Post Titer of TNF Specific Implantation Antibody in vivo 0none detected 1 10 2 30 6 70 8 100  11  60 15  23A control immunoisolatory vehicle maintained in vitro exhibited similarantibody release.

EXAMPLE 16 Subcutaneous Implantation of Encapsulated Islets Preparationof Islet Containing Capsules

Two types of acrylic copolymer hollow fibers, designated Type 1 and Type2 fibers, were used. Fibers were formed by using a dry-wet spinningtechnique with a spinneret as described in Cabasso, Hollow FiberMembranes, vol. 12, Kirk-Othmer Encyclopedia of Chemical Technology,Wiley, N.Y., 3rd Ed., pp. 492-517 (1980). The acrylic co-polymer usedwas poly(acrylonitrile-co-vinyl chloride)(M_(n)=100,000, M_(w)=300,000as measured by size-exclusion chroma-tography; CytoTherapeutics, Inc.)dissolved in dimethyl sulfoxide (12.5% w/w). The acrylic copolymersolution was pumped through the outer tube of the spinneret and waterwas pumped through the inner tube. Type 1 hollow fibers were extrudedinto water through an air gap, resulting in a fenestrated outer walltypical of fibers made by a dry-wet spinning technique. The type 2fibers were made in a analogous fashion, except the air gap was replacedby a humidified atmosphere, resulting in a smooth outer surface.

Rat islets were isolated from male Wistar-Furth rats as described inExample 1 above. The islets were immobilized in alginate gel andencapsulated into 2-cm type 1 or type 2 fibers, 550 or 1000 islets perfiber, as described in Lacy, P. E., et al., Science, 254, p. 1782(1991).

Implantation

The encapsulated rat islets were implanted intraperitoneally orsubcutaneously in mice made diabetic by the injection of streptozotocin.Non-fasting plasma glucose concentrations were determined three timesweekly; the diabetic recipients had concentrations greater than 400mg/dl before transplantation. The loading density was 70 islets percentimeter for 1000 islets and 35 islets per centimeter for 500 islets.Twenty-six mice received fibers intraperitoneally, and 26 receivedfibers subcutaneously. In each group, 14 mice received fibers containinga total of 1,000 islets and 12 received fibers containing a total of 500islets.

Results

The intraperitoneal type 1 fibers induced and maintained normoglycemiafor greater than 60 days in seven of nine recipients that received 1000islets and in all of the recipients receiving 500 islets. None of therecipients of subcutaneous type 1 implants of 500 islets remainednormoglycemic for 60 days; three of eight recipients of 1000 islets werenormoglycemic for greater than 60 days. Removal of the fibers from thesethree recipients returned the mice to a diabetic state. Transplants ofrat islets in the type 2 fibers produced and maintained normoglycemia inthe recipient mice in greater than 80% of either the intraperitoneal orsubcutaneous sites with either 1000 or 500 islets. The recipients becamehyperglycemic again when the fibers were removed at 60 days. Histologicexamination of the removed type 1 and type 2 fibers revealed that theywere biocompatible.

EXAMPLE 17 Controlled Reaggregation of Rat Islets

Rat islets were isolated, dissociated, reaggregated and encapsulated asdescribed in example 11, with the exception that sealed fibers were notexposed to serum in vitro and were held for only 1 hour prior toimplantation. Either two 2 cm long capsules or two 2 cm and one 1 cmcapsules were implanted in each rat.

Islets were dissociated and reaggregated to an approximate size of 35um. All the reaggregated cells from approximately 500 islets were loadedinto each 4-6 cm length capsule. Implanted capsules are capable ofmaintaining normoglycemia in rats for greater than 60 days.

EXAMPLE 18 Fabrication of Flatsheet Islet Encapsulation Vehicles

Aseptic materials and methods were used in all the following procedures.This included autoclave sterilization, EtOH sanitization, UVsterilization and/or 0.2 um sterile filtration.

Casting solution was prepared using a monoacrylic copolymer with anaverage molecular weight of 10⁵ daltons which was dissolved in awater-soluble, organic solvent. The casting solution was 10.0% w/wpolymer in the organic solvent. The polymer was precipitated once understerile conditions prior to its use to remove any residual monomers,oligomers, or any additives placed in the bulk polymer by themanufacturer. This polymer solution was then dried and redissolved in100% DMSO to form a 10% w/w polymer solution. This solution was passedthrough a 0.2 um sterile nylon filter and collected under asepticconditions.

Next the casting solution was uniformly spread using a casting bar overa ¼″ glass substrate at a casting thickness of 125 um. In order to castthe film, the substrate, held at a 30 degree incline, was moved underthe stationary casting bar into the precipitation bath. The level of theprecipitation bath was between ⅛-¼″ from the casting bar. The substratecan be any material which prevents premature lifting of the membranefrom the substrate prior to complete precipitation. Simultaneous withspreading, the casting solution was plunged into 24° C. water resultingin precipitation of the polymer forming an anisotropic semipermeablemembrane, with the permselective layer appearing as a thin skin on thequench side (away from substrate) of the membrane. The film was left inthe bath for four minutes to insure that membrane properties have beenadequately established prior to its removal.

The film was then carried through a series of rinses to remove anyresidual solvent or toxic residue that may compromise the compatibilityof the final product. These rinse baths were composed of solutions thatcaused no marked physical or chemical modification to the initialmembrane. The first post bath consists of water processed through aMilli-Q purification system and was left to soak for a minimum of 15min. The material was then removed and placed into a 70% v/v punctiliousethyl alcohol and water solution, which had been 0.2 um filtered, for aminimum of 60 mins and then removed. The final stage was soaking of thefilm in two sequential sterile normal saline baths with volumes of 2ml/cm² fiber for a minimum of 60 min each.

Results

The final wet-as-cast membrane thickness was between 30 um to 75 um witha hydraulic permeability of 0.475 cc/min/cm² at 5.0 psig. Rejectioncoefficient data indicated that the membrane excluded-substances largerthan approximately 100,000 daltons.

Encapsulation and Implantation

Sterile, cast 10% flatsheet membrane soaking in saline was prepared asdescribed above, and encapsulated islets were prepared as follows: A 6by 8 inch sheet of wet membrane was folded over on itself with the skinsides facing so as to create a strip of double membrane about 1″ wide.The fold was carefully pressed to form a crease. Using a #10 scalpelblade, the 1″ wide strip of double membrane was cut off from the rest ofthe sheet. The double strip was picked up by one end and cut into 1″squares using scissors. The squares were caught in saline as they werecut off. The tip and bottom of each square were connected by the foldcomprising one side.

Immediately prior to loading, a square was lifted out and placed on thelid of a 3″ diameter polystyrene petri dish previously wet with 1-2 mlof 1% CaCl₂ solution. The membrane was unfolded and each side wasfloated on the CaCl₂ solution with care taken to assure that thesolution did not flow into the tip of the membrane.

Previous to this, islets were allowed to settle into a pellet, andresuspended in 1% ungelled sodium alginate solution (solution wasprepared by making a 2% alginate solution in H₂O and mixing this 50/50with medium in which islets were cultured). Islet/alginate slurry wasgently mixed by stirring and aspirating. Slurry was prepared at the rateof 500 islets in 25 ul alginate per sq cm useable surface area on asingle membrane side. This equates to approximately 125 ul and 2500 ratislets for a 1″ sq membrane sheet.

Islet/alginate slurry was aspirated into a 200 ul pipet tip and thenevenly spread across the insid of one membrane leaving about 18 inch gapalong all edges of the square sheet. Spreading was done rapidly asalginate slowly crosslinked due to Ca⁺⁺ diffusion through the membranefrom the underlying CaCl₂ solution.

Once the alginate was sufficiently crosslinked to prevent alginatesmearing (approx 1-2 min) the other side of the flat sheet device wasfolded over to form a tip to the flatsheet sandwich. Care was taken toeliminate air bubbles.

The two sides of the membrane were sealed using an impulse heat sealerwith a ⅛″ heating element set on medium heat (temp reached between80-160° C.). Each edge, including the folded one, was individuallysealed by activating the heat sealer while pressing down on the ⅛″ stripof non-alginate coated membrane along each edge.

After sealing, the device was soaked in 1% CaCl₂ solution for 4 min soas to further crosslink the alginate. The device was held in Hank'ssolution until implantation (within 2 hours).

Flat sheet was implanted in the peritoneal cavity of a chemicallydiabetic recipient Wistar-Furth rat by making a midline incision throughthe skin and into the peritoneal cavity of the anesthetized rat. Theflat sheet implant was placed in the cavity by grasping the sealed edgewith smooth forceps and gently laying the device, free floating on topof the gut pile proximal to the peritoneal wall. The peritoneal cavityand skin were closed by suturing.

Animals were studied for 21 days, at which time the devices wereexplanted. Blood glucose levels dropped from 375 mg/dl to 150 mg/dlwithin 4 days of implant and remained there until explant, at which timeglucose values rose to 275 mg/dl (n=2).

Histological examination revealed viable islets immobilized in thealginate layer with less than a monolayer of cells attached to theoutside of the membrane.

EXAMPLE 19 Implantation of Encapsulated Bovine Adrenal Chromaffin Cellsin the Lumbar Subarachnoid Space in Sheep

Adrenal Gland Harvesting

Bovine adrenal chromaffin cells were obtained from healthy livestocksources in herds tested for adventitious agents and known to be free ofbovine Spongiform Enchephalitis. Two to three week old calves weighing52-72 kg (62±7) were premedicated with atropine (100 mg/kg) and xylazinechlorhydrate (0.15 mg/kg). Anesthesia was induced with pentobarbitalsodium (8 mg/kg), and maintained with 0.5-1% halothane delivered throughan endotracheal tube. The aorta and vena cava were isolated through acruciate organ harvesting ventral incision. The distal aorta and venacava, the coeliac axis, and the superior and inferior mesentericarteries were ligated. The proximal vena cava was clamped above theliver, and the proximal aorta above the coeliac axis. Four to six litersof cold saline solution and 2 liters of a hospital prepared organpreservation solution (formulated as the University of Wisconsinsolution minus hydroxyethylstarch and adenosine) were perfused by acannula introduced in the aorta. The organ preservation solutioncomprises potassium lactobionate 100 mM, KH₂PO₄ 25 mM, MgS04 5 mM,raffinose 30 mM, glutathion 3 mM and allopurinol 1 mM. The adrenalglands were then harvested with their native vessels and placed in asterile container filled with enough organ preservation solution tocover the gland. The container was placed on ice and send to the tissueculture laboratory for isolation of the chromaffin cells. Asepticsurgical techniques were utilized for all procedures. All harvestedglands were of suitable quality for subsequent chromaffin cellisolation. The total amount of cells obtained by the isolation techniqueranged from 2.0 to 3.0×10⁷ cells per gland with viability greater than95%, as assessed with FDA and PI stain. Cells were typically organizedin clusters of 50 to 200 μm in diameter.

Chromaffin Cells Isolation and Culture

A cannula was inserted into the isolated gland through the suprarenalvein. The glands were then perfused with 10 ml of cold organpreservation solution via the cannula until clear perfusate was seendripping from the gland. Five to seven ml of a 0.2% collagenase solution(Sigma, Type H) was then injected into each gland. The vein was clampedand the glands were placed in sterile beakers containing 100 ml of organpreservation solution and shaken in a 37° C. waterbath at 1 Hz for 30minutes. This first digestion allowed mechanical separation of thecortex from the medulla by gentle pulling. The medullary tissue was thenplaced in a tissue culture dish with 1 ml of organ preservation solutionand chopped into approximately 1-2 mm² sections. The chopped tissuepieces were then poured into a dissociation-filtration chamber with a250 μm pore size filtration grid, filled with 10 ml of 0.2% collagenasesolution and agitated at 1 Hz for 10 minutes at 37° C. Constanttemperature in the chamber was maintained by an external water jacket.Every 10 minutes the chamber was rinsed with cold organ preservationsolution. The isolated cells and cell clumps which passed through thefiltration grid were collected in a sterile 50 ml conical tube. A totalof three digestions were generally needed to fully digest the medullarytissue.

The tubes were then spun at 800 g for 5 minutes and washed twoadditional times with the organ preservation solution. An aliquot of thefinal wash supernatant was placed in thioglycolate medium for sterilitytesting.

The cells from each tube was plated in separate 100 mm tissue culturedishes, in 10 ml of PC-1 media, a defined medium containing protein fromhuman recombinant sources (Hycor Biomedical Inc.).

Using a 10 mL pipet, cells were removed from each petri dish and werepooled in a 50 ml centrifuge tube. The cell solution was spun at 800 gat ambient temperature for 5 minutes. The supernatant was disposed andthe pellet resuspended in 10 mL of HEPES buffered NaCl. Two 50microliter aliquots of cell suspension were placed in separate Eppendorftubes. One mL HEPES buffered NaCl and 1 mL FDA-PI were added to onetube, and 50 microliters Trypan blue solutions and 50 microliters Tritonx-100 were added to the other tube. The Triton-Trypan cell suspensionwas examined on a hemocytomer and the cells were counted. The number ofcells was found to be approximately 2-3×10⁶ per mL. The FDA-PI cellsolution was examined under a fluorescent microscope and the percentageof live cells was found to be greater than 95%.

The tissue culture dishes were held in a 5% CO₂ incubator at 37° C.,until the cells were loaded into capsules.

Preparation of Alginate Solution

A 2% alginate solution was prepared dissolving 1 g of Protan Ultrapurealginate which had been cold cycle ETO sterilized in 50 mL of HEPESbuffered 0.9% NaCl. The cell solution was diluted in the ratio of twoparts alginate solution to one part cell solution.

Encapsulation Procedure

Hollow fibers were spun from a 12.5-13.5% poly(acrylonitrilevinylchloride) solution by a wet spinning technique. Cabasso, HollowFiber Membranes, vol. 12, Kirk-Othmer Encyclopedia of ChemicalTechnology, Wiley, N.Y., 3rd Ed. pp. 492-517 (1980). The resultinghollow fiber had an outside diameter (OD) of around 900 μm and a wallthickness of around 150 μm. The fibers had a hydraulic permeability of18 ml/min/m²/mmHg and a rejection coefficient of more than 90% forbovine serum albumin. Fibers were impregnated with glycerine for storagepurposes.

In order to make implantable capsules, lengths of fiber were first cutinto 5 cm long segments and the distal extremity of each segment wassealed with an acrylic glue. Encapsulation hub assemblies were preparedby providing lengths of the membrane described above, sealing one end ofthe fiber with a single drop of LCM 24 (Light curable acrylate glue,available from ICI), and curing the glue with blue light, and repeatingthe step with a second drop. The opposite end was previously attached toa frangible necked hub assembly, having a silicone septum through whichthe cell solution may be introduced. The fiber was glued to the hubassembly by applying LCM 22 to the outer diameter of the hub assembly,and pulling the fiber up over it, and curing with blue light. Thehub/fiber assemblies were placed in sterilization bags and were ETOsterilized.

Following sterilization with ethylene oxide and outgassing, the fiberswere deglycerinated by ultrafiltering first 70% EtOH, and then HEPESbuffered saline solution through the walls of the fiber under vacuum.

The cell/alginate suspension (approx. 20×10⁶ cells/100 μl) was placed ina 1 ml syringe. A Hamilton 1800 Series 50 microliter syringe was set fora 15 microliter air bubble, and was inserted into a 1 ml syringecontaining the cell solution and 30 microliters were drawn up. The cellsolution was injected through the silicone seal of the hub/fiberassembly into the lumen of a modacrylic hollow fiber membrane with amolecular weight cutoff of approximately 50,000 daltons. Ultrafiltrationcould be observed along the entire length of the fiber. After oneminute, the hub was snapped off the sub-hub, exposing a fresh surface,unwet by cell solution. A single drop of LCM 24 was applied and theadhesive was cured with blue light. The device was placed first in HEPESbuffered NaCl solution and then in CaCl₂ solution for five minutes tocross-link the alginate. Each implant was about 5 cm long, 1 mm indiameter, and contained approximately 2.5 million cells.

After the devices were filled and sealed a silicone tether (SpecialitySilcone Fabrication, Paso Robles, Calif.) (ID: 0.69, OD: 1.25) was thenplaced over the proximal end of the fiber. A radiopaque titanium plugwas inserted in the lumen of the silicone tether to act as aradiographic marker. The devices were then placed in 100 mm tissueculture dishes in 1.5 ml PC-1 medium, and stored at 37° C., in a 5% CO₂incubator for in vitro analysis and for storage until implantation.

Implantation

Devices were implanted one week following the cell loading procedure.Sheep weighing 42-90 kg (69±15) were given general, endotrachealanesthesia (pentobarbital sodium 10 mg/kg iv; halothane 0.5-2%) andpreoperative antibiotics (cefazolin sodium 1 g iv). The animals werepositioned in the prone position and the operating table tilted head upat 30°. A 510 cm parasaggital lumbar incision was made and a spinal tapperformed with a Tuohy needle between L4 and L5 via an obliqueparamedian approach. The appropriate position of the needle in thesubarachnoid space was confirmed by withdrawal of several mls of CSF.This CSF was analyzed for cell counts, protein level, and microbiology.A guide wire was introduced through the lumen of the Tuohy needle untilit extended 4-5 cm cranially from the needle opening. The Tuohy needlewas removed and a 7 French dilator introduced over the guide wire to thelevel of the dura and removed, enlarging the wire track through thefascia, paraspinous muscle and ligamentum flavum. This allowed a 6French dilator with a 20 cm long outer cannula sheath to be advancedinto the subarachnoid space until the tip of the cannula was positioned7 cm within the space. The guide wire and the dilator were then removed,leaving the cannula within the subarachnoid space to act as a protectiveguide for insertion of the encapsule.

The cell-loaded and fully assembled device was delivered into theoperating room in a sterile container, bathed in PC-1 medium. The devicewas prepared for insertion by mounting the tether on a stainless steelpusher which served to stiffen the very flexible tether and allowed thecapsule to be manipulated within the lumen of the cannula. The membraneportion of the device was then introduced into the cannula, handling thedevice by the silicone tether and the handle of the pusher. The devicewas advanced until the membrane portion lay entirely within the CSFcontaining subarachnoid space. The cannula was then removed while thedevice was maintained in position using the pusher. Finally, the pusherwas removed and the silicone tether anchored at its free end by anon-absorbable suture and completely covered with a 2 layer closure ofskin and subcutaneous tissue.

The animal was recovered, examined for possible neurologicalcomplications, and returned to the farm for boarding on the day ofimplantation. All animals were able to return to normal diet andactivity on the day of surgery. All experimental, animal care andsurgical protocols were approved by the Canton of Vaud Committee onanimal research.

Explantation

Four to eight weeks post-implantation each sheep was anesthetized asdescribed above. The subcutaneous portion of the silicone tether wasisolated through a small skin issue incision. The device was thenretrieved by gentle traction. The capsule was placed in PC-1 media foranalysis of catecholamine release and then fixed in 4% paraformaldehydesolution for histology. A spinal tap was performed for all cell counts,protein level and microbiology prior to the removal of the device. Theanimal was allowed to recover and one week following retrieval of thedevice, CSF samples were again taken and the animal was sacrificed byoverdosage of pentobarbitral.

Neurochemical Assays

The ability of the capsules to release catecholamines was determinedbefore and after transplantation. Each capsule was first placed in 2 mlHank's balanced buffered saline (HBSS) solution for 30 minutes and basalrelease samples were collected. Evoked release was obtained byincubating the capsule in 63 μm nicotine solution in HBSS for another 30minutes. Perchloric acid (1N) was added to the collected samples as anantioxidant. Catecholamines levels were determined by reverse phase highperformance liquid chromatography (HPLC) with electrochemical detection.

Histology

Following fixation in 4% paraformaldehyde, the retrieved capsules wererinsed with phosphate buffered saline (PBS), dehydrated in gradedalcohol up to 95% and embedded in blycol methacrylate infiltrationsolution (Historesine Mounting Medium, Reichert-Jung). Three micronthick sections were cut on a microtome (Supercut 2065, Leica), mountedon glass slides and stained with cresyl violet. For immunohistochemistrythe capsules were also fixed in 4% paraformaldehyde, embedded in 5%agarose and cut on a cryostat (Cryocut 1800, Leica). The immunostainconsisted of a mouse monoclonal antibody to tyrosine hydroxylase(Boehringer Mannheim) using the peroxidase-anti-peroxidase (PAP)technique and diaminobenzidine (DAB) coloration.

Results

Neurochemical Analysis

All capsules released a significant amount of catecholamines undernicotine stimulation. An increase in the catecholamine release from thecapsules on day-1 (one day before implantation, seven dayspost-isolation) was observed with each subsequent isolation andencapsulation series. The mean evoked release of each batch ranged from362±14 to 1464±300 pmol/2 ml/30 min for norepinephrine and 161±11 to1350±344 pmol/2 ml/30 min for epinephrin. As indicated by the standarddeviation, there were small but noticeable variations in catecholaminerelease between the various capsules of each batch. Basal release wasbelow 230 pmol/2 ml/30 min for both catecholamines measured.

Typically, the cohorts maintained in vitro were analyzed at day-1, +7,+14, +21 and +28 days following transplantation for evoked release ofcatecholamines. All capsules continued to respond to nicotinestimulation for at least one month post-encapsulation. Some showed anincrease in their release over time (batch 1,2), some remained stable(batch 3), and some demonstrated a progressive decrease of their releaseover time (batch 4, 5).

Histology

Microscopic examination showed good viability of the encapsulated cells.The cells were organized in small aggregates entrapped in the alginatematrix. These cell clusters were all positive for tyrosine-hydroxylaseimmunochemistry. There was some disparity in capsule loading within andbetween batches.

Surgery and Behavior

No infections were observed in the implanted sheep and all CSF samplescollected were sterile. No increase in leucocyte counts were observed inthe CSF between the implantation and explantation times. The same wastrue for protein levels with exception of sheep 5 which showed adoubling of CSF protein concentration at the explantation time. Atraumatic spinal tap at the explantation may explain this increase. Ofthe six implanted sheep, two showed a transitory weakness of the hindlimbs following the transplantation procedure. An additional sheepshowed a complete paralysis at the time of recovery and did not show anyimprovement in the following hours. This animal was sacrificed on thefirst day post-transplantation and was therefore not included in thepresent series. At autopsy the device appeared to have perforated thespinal cord of the animal.

No further surgical complications were encountered. All devices wereretrieved through a small skin incision by gentle traction on the tetherat 4 or 8 weeks post-implantation. The silicone tethers remained firmlyfixed to the capsules; the membranes remained integral and attached tothe tether.

Morphologic Analysis

All the retrieved capsules were intact on gross examination. Themembrane was devoid of host cells by microscopic examination. Clustersof viable cells dispersed in the alginate matrix were observedthroughout the capsule. The cell aggregates were strongly positive fortyrosine hydroxylase. Capsule loading varied between batches, with ageneral upward trend.

Release

After retrieval, explanted devices were tested for catecholamine releasein order to assess chromaffin cell viability and responsiveness tonicotine stimulation. Basal and stimulated release levels were measuredand compared to levels in in-vitro cohorts. With the exception of sheep3, the evoked release of retrieved capsules was in the same range astheir respective in vitro cohort evoked release. In sheep 4, the releaseof the retrieved capsule was higher than that of its in vitro cohort.For Sheep 6, both explanted capsules and in-vitro controls had lowlevels of catecholamine release.

EXAMPLE 20 Implantation of Encapsulated Cellular Grafts in the LumbarSubarachnoid Space in Humans

Fiber Characteristics

The semipermeable membrane fibers used in this trial were double skinnedPAN/PVC fibers having the following dimensions: an inner diameter of 773microns, an outer diameter of 920 microns, and a wall thickness of 73.1microns.

Preparation and Encapsulation of Calf Adrenal Cells

Bovine adrenal chromaffin cells were prepared and encapsulated asoutlined in example 19.

Surgical Procedure

After establishing IV access and administering prophylactic antibiotics(cefazolin sodium, 1 gram IV), the patient was positioned on theoperating table, generally in either the lateral decubitus orgenu-pectoral position, with the lumbar spine flexed anteriorly. Theoperative field was sterily prepared and draped exposing the midlinedorsal lumbar region from the levels of S-1 to L-1, and allowing forintraoperative imaging of the lumbar spine with C-arm fluoroscopy. Localinfiltration with 1.0% lidocaine was used to establish anesthesia of theskin as well as the periosteum and other deep connective tissuestructures down to and including the ligamentum flavum.

A 3-5 cm skin incision was made in the parasagital plane 1-2 cm to theright or left of the midline and was continued down to the lumbodorsalfascia using electrocautery for hemostasis. Using traditional bonylandmarks including the iliac crests and the lumbar spinous processes,as well as fluoroscopic guidance, and 18 gauge Touhy needle wasintroduced into the subarachnoid space between L-3 and L-4 via anoblique paramedian approach. The needle was directed so that it enteredthe space at a shallow, superiorly directed angle that was no greaterthan 30-35° with respect to the spinal cord in either the sagittal ortransverse plane. Appropriate position of the tip of the needle wasconfirmed by withdrawal of several ml of cerebrospinal fluid (CSF) forpreimplantation catecholamine, enkephalin, glucose, and protein levelsand cell counts.

The Touhy needle hub was reexamined to confirm that the opening at thetip is oriented superiorly (opening direction is marked by the indexingnotch for the obturator on the needle hub), and the guide wire waspassed down the lumen of the needle until it extended 4-5 cm into thesubarachnoid space (determined by premeasuring). Care was taken duringpassage of the wire that there was not resistance to advancement of thewire out of the needle and that the patient did not complain ofsignificant neurogenic symptoms, either of which observations mightindicate misdirection of the guide wire and possible impending nerveroot or spinal cord injury.

After the guide wire appeared to be appropriately placed in thesubarachnoid space, the Touhy needle was separately withdrawn andremoved from the wire. The position of the wire in the midline of thespinal canal, anterior to the expected location of the caud equina, andwithout kinks or unexplainable bends was then confirmed withfluoroscopy. After removal of the Touhy needle the guide wire should beable to be moved freely into and out of the space with only very slightresistance due to the rough surface of the wire running through thedense and fibrous ligamentum flavum.

The 7 French dilator was then placed over the guide wire and the wirewas used to direct the dilator as it was gently but firmly pushedthrough the fascia, paraspinous muscle, and ligamentum flavum, followingthe track of the wire toward the subarachnoid space. Advancement of the7 French dilator was stopped and the dilator removed from the wire assoon as a loss of resistance was detected after passing the ligamentumflavum. This was done in order to avoid advancing and manipulating thisrelatively rigid dilator within the subarachnoid space to anysignificant degree.

After the wire track was “overdilated” by the 7 French dilator, the 6French dilator and cannula sheath were assembled and placed over theguide wire. The 6 French dilator and cannula were advanced carefullyinto the subarachnoid space until the opening tip of the cannula waspositioned 7 cm within the space. As with the 7 French dilator, theassembled 6 French dilator and cannula were directed by the wire withinthe lumen of the dilator. Position within the subarachnoid space wasdetermined by premeasuring the device and was grossly confirmed byfluoroscopy. Great care was taken with manipulation of the dilators andcannula within the subarachnoid space to avoid misdirection and possibleneurologic injury.

When appropriate positioning of the cannula was assured, the guide wireand the 6 French dilator were gently removed from the lumen of thecannula in sequence. Depending on the patient's position on theoperating table, CSF flow through the cannula at this point should benoticeable and may be very brisk, requiring capping the cannula or veryprompt placement of the capsule implant in order to prevent excessiveCSF.

The encapsulated adrenal chromaffin cell graft (CytoTherapeuticsCereCRIB™) was provided in a sterile, double envelope container, bathedin transport medium, and fully assembled including a tubular siliconetether. Prior to implantation through the cannula and into thesubarachnoid space, the capsule was transferred to the insertion kittray where it was positioned in a location that allowed the capsule tobe maintained in transport medium while it was grossly examined fordamage or major defects, and while the silicone tether was trimmed,adjusting its length to the pusher and removing the hemaclip™ that plugsits external end.

The tether portion of the CereCRIB™ capsule was mounted onto thestainless steel pusher by inserting the small diameter wire portion ofthe pusher as the membrane portion of the device was carefullyintroduced into the cannula. The capsule was advanced until the tip ofthe membrane reached a point that was 2-10 mm within the cranial tip ofthe cannula in the subarachnoid space. This placement was achieved bypremeasuring the cannula and the capsule-tether-pusher assembly, and itassured that the membrane portion of the capsule was protected by thecannula for the entire time that it was being advanced into position.

After the capsule was positioned within the cannula, the pusher was usedto hold the capsule in position (without advancing or withdrawing) inthe subarachnoid space while the cannula was completely withdrawn fromover the capsule and pusher. The pusher was then removed from thecapsule by sliding its wire portion out of the silicone tether. Usingthis method the final placement of the capsule was such that the 5 cmlong membrane portion of the device lay entirely within the CSFcontaining subarachnoid space ventral to the cauda equina. It wasanchored at its caudal end by a roughly 1-2 cm length of silicone tetherthat ran within the subarachnoid space before the tether exited throughthe dura and ligamentum flavum. The tether continued externally fromthis level through the paraspinous muscle and emerged from thelumbodorsal fascia leaving generally 10-12 cm of free tether materialthat was available for securing the device.

CSF leakage was minimized by injecting fibrin glue (Tissel®) into thetrack occupied by the tether in the paraspinous muscle, and by firmlyclosing the superficial fascial opening of the track with a purse-stringsuture. The free end of the tether was then anchored with non-absorbablesuture and completely covered with a 2 layer closure of the skin andsubcutaneous tissue.

The patient was then transferred to the neurosurgical recovery area andkept at strict bed rest, recumbent, for 24 hours postoperatively.Antibiotic prophylaxis is also continued for 24 hours following theimplantation procedure.

Human Pain Patients

Three human terminally ill patients suffering from intractable pain wereimplanted according to the method outlined above.

Devices were released for implantation only after individual testing forsterility and for release of catecholamines. The protocol called for athirty day study that could be extended to a maximum of 90 days upon therequest of the patients. Three patients were eligible with terminalcancer, pain incompletely relieved by narcotic therapy, and no evidenceof active infection or tumor in the meningeal space. After informedconsent was granted by the patients and approval was received from theEthical Committee of the Faculty of Medicine of the University ofLausanne, Switzerland, the devices were implanted under localanesthesia.

Postoperative recovery was uneventful though all patients experiencedsome loss of CSF fluid and one patient experienced headaches of severaldays duration. Two of the three patients recorded improvement on theMcGill questionnaire and a visual analog scale of pain; the third didnot. Significant increases were observed in the cerebrospinal fluidcatecholamine levels of the two patients with improved pain scores. Allthree patients reduced their intake of narcotics and analgesics (Table3).

The tethered implants were recovered via simple surgical excision after43 days and 55 days in two patients and at autopsy in patient #3 whodied from her primary disease at day 42. Explanted devices wereinspected visually and then examined histologically and for biochemicalactivity. There was no visible difference between the devices asimplanted and as retrieved. Upon microcoscopic examination, externalsurfaces of all three implants were free of adherent cells, fibroticoverlayers, and other signs of acute phase response or foreign bodyreaction.

Intracapsular populations of healthy chromaffin cells were observed byhistology in all three explants, with cell viability estimated at 80percent. Cells recovered from the capsules were also positive byimmunohistochemistry for tyrosine hydroxylase and metenkaphalins. Basalrelease of norepinephrine and epinephrine in explanted capsules was inthe range of 0.2 and 3 nanomoles per 24 hours. Autopsy reports on thespinal cords became available in all three patients and showed no effectfrom the implant.

TABLE 3 MORPHINE INTAKE (mg/day) pre-implant post implant¹ oral Patient1 60 0 Patient 2 0 0 Patient 3 60 0 epidural Patient 1 75 18 Patient 260 32 Patient 3 0 0 ¹Mean value from day 10 post implant to explant (ordeath)

EXAMPLE 21 Implantation of Encapsulated Cellular Grafts Intracranially,in the Lateral Ventricle in Humans

Two human patients suffering from intractable pain were implanted in theventricle of the brain with encapsulated adrenal chromaffin cells. Thebrain ventricles, including the lateral ventricles, lie rostral to thelumbar region. The CSF drains or flows from the brain to the spinalcord. The chromaffin cells and CereCRIB™ capsules were prepared asdescribed in Example 20.

The surgical procedure for implantation into the lateral ventricle ofthe brain is described below.

Immediately before the implantation procedure, the patient was fittedwith a stereotactic head ring assembly and localizer ring (or imagelocalization/marker device) suitable for guided cannula placement withinthe lateral ventricles using local anesthesia (local infiltration withgenerally 1% lidocaine). The Radionics® BRW frame was used here, howeverthe Radionics® CRW, Leksell®, or functionally similar devices are alsoappropriate.

A computed tomography (CT) scan was then performed and used to define atarget site(s) and stereotactic coordinates for the implant(s).Implantation cannula trajectory and implant site were chosen with thefollowing considerations: (1) avoiding the frontal sinuses; (2) avoidingthe choroid plexus; (3) allowing straight, undistorted positioning ofthe intended implant within the lateral ventricle. There are threecapsule lengths, 2.5, 3.75, or 5.0 cm, currently in use. The twopatients in this study were implanted with 2.5 cm CereCRIB™ capsules.

A target site must be selected that will allow a length of the internalend of the cannula that is at least the length of the membrane portionof the desired capsule to lie within an acceptable, CSF filled spacewithin the ventricle. The zero reference point for determining cannulainsertion depth is the surface of the skin, as seen on the CT scan, andthe target site is defined as the intended target of the internal tip(opening) of the insertion cannula.

Two implant devices may be placed in one patient at a single procedureby placing one implant in each lateral ventricle. Future implantationsites may target the third ventricle and/or the aqueduct. The currentstereotactic guidance technique uses CT imaging for reference, howevermagnetic resonance imaging (MRI), stereotactic at last coordinates,ultrasound or other guidance methods may also be appropriate. Followingcompletion of the data gathering for stereotactic placement of theimplant(s), the patient is transferred to the operating room for theimplantation procedure.

After establishing IV access and administering prophylactic antibiotics(currently, cefazolin sodium, 1 gram IV), the patient was positioned onthe operating table in the semi-supine/seated position with thestereotactic head ring assembly secured to the table. The operativefield was sterily prepared and draped exposing the intended implantationsite(s) (generally located in the paramedian, frontal region) andallowing for sterile placement and removal of the stereotactic arcsystem/manipulator to the frame base.

Local infiltration with 1.0% lidocaine was used for anesthesia of theskin and deeper scalp structures down to the periosteum, and a 4-8 cmskin incision was made down to the skull at the calculated entry site(s)for the stereotactically guided insertion canula (generally in thefrontal region, in the parasagital plane 3 cm to the right or left ofthe midline) using electrocautery for hemostasis. A twist drill guidedby the stereotactic arc system was then used to create a burr hole(generally 4 mm diameter) down to the level of the dura. The dura wassharply penetrated, and the insertion cannula/obturator assembly wasmounted into the stereotaxic microdrive and directed into the burr hole.Blood from the wound was excluded from the burr hole by applying themicrodrive guide tube directly against the rim of the burr hole.

The insertion cannula/obturator assembly were advanced manually to thepreset depth stop on the microdrive, leaving the tip of the cannula atthe target site. The obturator was then carefully withdrawn from theinsertion cannula, taking care not to deflect the cannula with the tipof the obturator. Appropriate position of the tip of the cannula withinthe ventricle may be confirmed by a meniscus of cerebrospinal fluid(CSF) rising up within the clear insertion cannula after removal of theobturator. Samples of CSF may be taken for preimplantationcatecholamine, enkephalin, glucose, and protein levels and cell counts.

The encapsulated adrenal chromaffin cell graft (CytoTherapeuticsCereCRIB™) was prepared and mounted onto the pusher as described inExample 20.

The CereCRIB™ capsule was handled completely by the silicone tether andthe handle of the pusher as the membrane portion of the device wascarefully introduced into the cannula. The capsule was advanced untilthe tip of the membrane reached a point that was within 1-2 mm of theinternal tip of the cannula positioned in the lateral ventricle (but notextending beyond the tip of the cannula). This placement was achieved bypremeasuring the cannula and the capsule-tether-pusher assembly, and itassured that the membrane portion of the capsule was protected by thecannula for the entire time that it was being advanced into position.After the capsule was positioned manually within the cannula, the pusherwas locked into position in the microdrive and used to hold the capsulein position in the ventricle (without advancing or withdrawing) whilethe cannula was completely withdrawn from over the capsule and pusher.The pusher was then removed from the capsule by sliding its wire portionout of the silicone tether.

Using this method the final placement of the capsule was such that theentire membrane portion of the device lay entirely within anappropriate, CSF containing region of the ventricle. The membranecapsule was anchored at its external end by a length of silicone tetherthat ran (generally) through a portion of the frontal lobe before itexited through the dura and the skull, leaving generally 5-10 cm of freetether material that was available for securing the device. The free endof the tether was then anchored to the outer table of the skull adjacentto the burr hole using a standard, maxillo-facial miniplate and screwsand completely covered with a 2 or 3 layer closure.

The patients were then transferred to the neurosurgical recovery areaand followed for 12 hours postoperatively for potential hemorrhagiccomplications with no special restrictions. Antibiotic prophylaxis wasalso continued for 24 hours following the implantation procedure.

EXAMPLE 22 Implantation of β-NGF Secreting BHK Cells for Prevention ofNeural Damage Due to Excitotoxicity (Huntington's Disease Model)

Subjects

Adult male Sprague-Dawley rats (Taconic Breeders, Germantown, N.Y.)approximately 3 months old and weighing 300-350 grams were used. Theanimals were housed in groups of 3 in a temperature andhumidity-controlled colony room which was maintained on a 12 hrlight/dark cycle with lights on at 0700 hrs. Food and water wereavailable ad libitum throughout the experiment.

BHK-NGF Cell Line Production

Two human genomic clones (phbeta N8D8, phbeta N8B9) coding for the 5′and 3′ ends of the β-NGF gene were obtained from the ATCC. A 440 bp 5′Sca1-EcoR1 fragment from phbeta N8D8 was ligated to a 3′ 2.0 kb EcoR1fragment isolated from phbeta N8B9. The spliced NGF genomic sequencecontained approximately 37 bp of the 3′ end of the first intron, thedouble ATG sequence believed to be the protein translation start forpre-pro-NGF and the complete coding sequence and entire 3′ untranslatedregion of the human gene (Hoyle et al., Neuron, 10, pp. 1019-34 (1993)).The combined 2.51 kb β-NGF fragment was subcloned into the DHFR-basedpNUT expression vector immediately downstream from the mousemetallothionein-1 promotor (−650 to +7) and the first intron of the rateinsulin II gene (Baetge et al., Proc. Natl. Acad. Sci., 83, pp. 5454-58(1986). The pNUT-βNGF construct was introduced into BHK cells using astandard calcium phosphate-mediated transfection method.Mock-transfected cells served as controls in these experiments. BHKcells were grown in DMEM, 10% fetal bovine serum,antibiotic/antimycotic, and L-glutamine (GIBCO) in 5% CO2 and at 37° C.Transfected BHK cells were selected in medium containing 200 μMmethotrexate (Sigma) for 3-4 weeks and resistant cells were maintainedas a polyclonal population either with or without 200 μM methotrexate.

Encapsulation Procedure

Asymmetric hollow fibers were cast from solutions of 12.5-13.5% poly(acrylonitrile vinyl chloride, PAN-PVC) copolymer in dimethyl sulfoxide(w/w). The fabrication process utilized a dry-wet (jet) spinningtechnique according to Cabasso, Hollow Fiber Membranes, vol. 12,Kirk-Othmer Encyclopedia of Chemical Technology, Wiley, N.Y., 3rd Ed.,pp. 492-517 (1980). Single cell suspensions of BHK cells were preparedusing calcium- and magnesium-free Hanks' balanced salts (HBSS) andtrypsin/EDTA. Encapsulation devices were manufactured by mounting a1-1.1 cm length of dry hollow fiber onto hub with a septal fixture atthe proximal end which has loading access for cells to be injected intothe lumen of the device. Cells were loaded into the prefabricatedcapsules as follows: BHK control cells and BHK/hNGF cells were loaded ata density of approximately 10⁷ cells/ml. The BHK cell suspensions, at adensity of 2×10⁷ cells/ml, were mixed 1:1 with physiologic Vitrogen®(Celtrix, Palo Alto, Calif.), and infused into the capsules through theseptal access port. After infusing 2.2.5 μl of the cellular suspension,the septum was cracked off and the access port was sealed using alight-cured acrylate (Luxtrak™ LCM 24, ICI Resins US, Wilmington,Mass.). The capsules were subsequently “tethered” by placing a 1.5 cm0.020″ silastic tube over the acrylic hub. The cell-loaded devices weretransferred into sterile 5 ml polypropylene snap cap tubes containing4.5 ml of PC-1 medium. The 5 ml snap cap tube was then placed inside asterile 50 ml conical centrifuge tube and sealed for transport.

BHK cell-loaded capsules were maintained in serum-free defined PC-1medium (Hycor Biomedical Inc., Portland, Me.) for 2-5 days prior toimplantation. After 3 or 4 days in vitro, cell-loaded capsules wererinsed in HBSS, placed in 1 ml of fresh PC-1 medium overnight, and themedium analyzed for hNGF by ELISA.

NGF ELISA

The quantification of hNGF released from encapsulated BHK/hNGF cells wasperformed as follows. All of the reagents were obtained fromBoehringer-Mannheim Biochemicals unless otherwise noted. Nunc-ImmunoMaxisorp ELISA plates were coated with 150 μl per well of anti-mouse-β(2.5S) NGF at 1 ng/ml in coating buffer (1×PBS without CaCl₂ and withoutMgClsub₂/0.1% sodium azide; pH 9.6). The coated plates were incubated at37° C. for at least 2 hours or alternatively at 4° C. overnight.

The coating solution was withdrawn from the wells and the wells werewashed 3× with 300 μl wash buffer (50 mm Tris-HCl/200 mm NaCl₂/1% TritonX-100/0.1% sodium azide; pH 7.0). The wells were then blocked with 300μl of coating solution containing 10 mg/ml of BSA at room temperaturefor 30 min. The wells were then washed 3× with 300 μl wash buffer.Conditioned medium samples were diluted 1:1 in 2× sample buffer (thesample buffer is the same as wash buffer, only with 2% BSA), with 10 μlof the prepared samples loaded into the wells. The plates were coveredand incubated for at least 2 hours at 37° C. or overnight at 4° C.

The solutions were removed from the wells by suction and washed 3× with300 μl of wash buffer. To each well, 100 μl of 4U/ml of anti-mouse-β(2.5S) NGF-β-gal conjugate was added. The plates were incubated at 37°C. for at least 1 hour. The solutions were removed from the wells bysuction and washed 3× with 300 of wash buffer. Finally, 200 μl ofchlorophenol red-β-D-galactopyranoside substrate solution (40 mg CPRG in100 mm Hepes/150 mm NaCl/2 mm MgCl₂/0.1% sodium azide/1% BSA; pH 7.0)was added to the wells, incubated at 37° C. for 30 min to one hr orafter the color development was sufficient for photometric determinationat 570 nm, with the samples analyzed on a plate reader and measuredagainst recombinant NGF protein standards.

Surgery

Immediately prior to surgery, rats were anesthetized with sodiumpentobarbital (45 mg/kg, i.p.), and positioned in a Kopf stereotaxicinstrument. A sagittal incision was made in the scalp and two holesdrilled for the placement of the cell-loaded capsules into the lateralventricle. Rats were unilaterally implanted by placing the capsulewithin an 18 gauge Teflon catheter mounted to the stereotaxic frame aspreviously described. The stereotaxic coordinates for implantation were:anterior 0.5 mm anterior to gregma, 1.5 mm lateral to the sagittalsuture and 8.0 mm below the cortical surface.

Three days following implantation of cell-loaded capsules, animals wereanesthetized, placed in the stereotaxic instrument and injected with 225nmol of quinolinic acid (“QA”) or phosphate-buffered saline (PBS) intothe striatum at the following coordinates: anterior++1.2 mm,lateral=±2.6 mm and ventral=−5.5 mm from the surface of the brain. QA(Sigma Chemical Co.) was dissolved in 2N sodium hydroxide and dilutedwith phosphate buffer at pH 7.2 to a final pH of 7.4 and concentrationof 225 nmol/ul. QA was infused into the striatum, using a 28-gaugeHamilton syringe, over five min in a volume of 1 μl. The injectioncannula was left in place for an additional two min to allow fordiffusion of the perfusate. This procedure resulted in the formation ofthree experimental groups: 1) quinolinic acid only (QA; N=8), quinolinicacid+NGF-secreting BHK cells (QA/BHK/hNGF; N=6), and quinolincacid+control BHK cells (QA/BHK/CONTROL; N=7).

Immediately following surgery, animals were injected i.p. with 10 ml oflactated Ringer's solution. Animals were housed postoperatively withfood mash and water available ad libitum.

At the conclusion of behavioral testing, animals were anesthetized andplaced into the stereotaxic instrument. A craniotomy was performed overthe implantation site and the dural scar surrounding the implant siteexcised. The cortical surface was cut to expose the underlying capsulewitch was retrieved with a pair of Dumont (#5) forceps.

Histology

A subset of animals (3-4 per group) were anesthetized 29-30 daysfollowing surgery and prepared for histological analysis. Animals weretranscardially perfused, using a peristaltic pump, with 20 ml salinefollowed by 500 ml of paraformaldehyde. All solutions were ice cold (4°C.) and prepared in 50 mM PBS (pH 7.4). Brains were removed followingfixation, placed in 25% buffered sucrose (pH 7.4) and refrigerated forapproximately 48 hr.

Tissue was cut at 40 μm intervals on a cryostat and mounted ontopolylysine coated slides. Alternating sections were taken throughout thestriatum and processed for immunocytochemical localization of cholineacetyltransferase (ChAT) and glial fibrillary acidic protein (GFAP)according to the following protocol: 1) overnight incubation in PBScontaining 0.8% Triton X-100+10% normal serum, 2) 48 hr incubation withprimary antibody (goat antiserum to ChAT; Chemicon) at a dilution of1:1000 or (rabbit antiserum to GFAP at a dilution of 1:5,000), 3) 6×5min rinses in PBS+0.2% Triton X-100 followed by a 1.5 hour incubation inbiotinylated secondary antibody (1 gG), 4) 6×5 min rinses in PBS+0.2%Triton X-100, 5) incubation with Avidin-Biotin Complex (ABC, VectorElite) for 1.5 hours, 6) 3×5 min rinses in PBS, 7) 5 min rinse indistilled water, 8) incubation DAB (0.05%)+2% nickel ammonium sulfate(ChAT only) dissolved in 0.1% Tris buffer for 5 min followed by hydrogenperoxide (0.01%) for 5 minutes, (9) the reaction was terminated by 3×1min rinses in PBS.

A separate series of sections throughout the striatum were stained forNADPH-diaphorase according to the procedure of Vincent et al. Adjacentsections were stained with hematoxylin and eosin (H+E). Sections weremounted, dehydrated and coverslipped.

For analysis of retrieved capsules, capsules were fixed in a 4%paraformaldehyde, 0.5% glutaraldehyde solution, rinsed in PBS anddehydrated up to 95% ethanol. A 1:1 solution of glycol methacrylate(Historesin, Reichert-Jung, Cambridge Instruments) was then added to thecapsules for one hr. Pure infiltration solution replaced the 1:1 mixtureand remained for a minimum of 2 hrs. The capsules were then rinsed withthe embedding solution, transferred to flat molds, and embedded inglycol methacrylate. Sections 5 μm thick were sectioned (Reichert-Jung,Supercut microtome 2065), mounted on glass slides and stained for H+E.

Behavioral Testing

Beginning 13-14 days following QA injections, animals were tested forapomorphine-induced (1.0 mg/kg in normal saline containing 0.2%ascorbate) rotation behavior in one of six rotation devices (Rotoscan,Omnitech Instruments) which were connected to an IBM computer forautomated data collection. Animals were placed into the test chamber fora 5 min habituation period and were then injected with apomorphine.Sensitization of apomorphine-induced rotation behavior occurs followingexcitotoxin lesions of the striatum. Therefore, animals were tested 4times with each session separated by a 3-4 day interval. Rotations weredefined as complete 360 degree ipsilateral turns and were reported asthe net difference between the two directions.

Statistical Analysis

The behavioral results were assessed using a two-way repeated measuresanalysis of variance (ANOVA). Appropriate pair-wise comparisons wereperformed using Fisher's Least Significant Difference test. Acceptablestatistical significance was established at p<0.05.

Results

Behavioral Testing

No overt signs of behavioral or neurological toxicity were observed inany animals following implantation of either BHK/hNGF or BHK controlcapsules. During the post-operative recovery period following QAinjections, the QA/BHK/CONTROL and QA alone groups exhibited whole bodybarrel rotations which persisted for 2-4 hours. These same animals had atransient period of weight loss, piloerection and diarrhea whichsubsided 3-4 days following QA. Animals which received QA together theBHK/hNGF capsules did not show whole body rotations but did exhibit aslight motor asymmetry following QA. This asymmetry was transient andrecovery was seen within several hrs. No additional signs of systemictoxicity were noted.

Following QA injections, animals displayed apomorphine-induced rotationsipsilateral to the lesion with the extent of rotation behaviorincreasing with repeated testing. hNGF treatment significantly decreasedthe extent of rotation behavior produced by QA. A two-way repeated ANOVArevealed significant effects of treatment F(2, 17)=16.063, p<0.0001, aswell as repeated testing F(2, 3)=28.861, p<0.0001, and a treatment bytesting interation F (6, 51)=2.937, p<0.05. Post-hoc analysis revealedthat the QA/hNGF group rotated significantly less than either the QAalone or QA/BHK control at all test times. No significant differenceswere observed between the QA alone and QA/BHK control groups at any timeduring testing.

NGF ELISA

Prior to implantation and following retrieval (immediately-prior toperfusion), the encapsulated BHK cells were incubated and theconditioned medium was assayed for hNGF by ELISA. Prior to implantation,the encapsulated BHK/hNGF cells were releasing 34.13 (±6.9) ngNGF/capsule/24 hr while BHK/CONTROL cells showed hNGF levels nodifferent than measured in control medium (0.06 ng NGF/capsule/24 hr).The BHK cell-loaded capsules were easily retrieved with little or nohost tissue adhering to the capsule wall. Post explant values of hNGFfrom capsules averaged 16.03 (±6.0) ng hNGF/capsule/24 hr and was notdetected in conditioned medium from BHK/CONTROL capsules.

Histology

The cell-loaded devices were easily retrieved and induced minimal damageto the host tissue. Placement of the capsules was within the lateralventricle in all cases. Analysis of sections throughout the implant siterevealed that the devices abutted the cortex and extended through thecorpus callosum to the ventral aspect of the lateral ventricle. Thecapsules typically extended into the host striatum and in some casesmedially into the lateral septum.

Administration of QA produced a substantial atrophy of the striatumtogether with a marked ventricular dilation. In some cases moderate cellloss was observed in the nucleus accumbens and cortical regions adjacentto the injection site. The lesion core was virtually devoid of neuronswith a nearly complete loss of ChAT and NADPH-d positive neurons. Ingeneral, the remaining neurons present within the lesion core wereshrunken and dystrophic in appearance. GFAP staining revealed thepresence of reactive astrocytes throughout the lesion site and extendinginto the adjacent host tissue.

Implantation of BHK control cell-loaded devices into the lateralventricles did not effect striatal morphology. Furthermore, noalterations in the size of the lesion core or extent of cell loss wasobserved in these animals. Infiltration of reactive astrocytes dominatedthe ventricular wall and the striatum immediately adjacent to theimplantation site. In contrast, implantation of encapsulated BHK cellswhich released hNGF exerted a marked protective effect on striatalmorphology following QA. In these animals, the lesion core wassignificantly reduced and frequently consisted of cell necrosissurrounding the injection tract with minimal extension into thesurrounding tissue. Within the lesion core there was no apparent sparingof neurons. On the other hand, NADPH-d, and Nissl staining revealed astriking preservation of neurons removed from the central lesion core.In general, the sparing of ChAT-positive neurons appeared greater thanthat of NADPH-d-positive neurons.

Capsule morphology and cell survival of the encapsulated BHK cells weredetermined. Few adhering host cells were found on the capsule surface.An abundance of viable BHK cells were evenly distributed throughout thecapsule. Areas of focal cell debris were occasionally observed withinthe cores of large viable cell aggregates. Numerous mitotic figures wereobserved throughout the capsules and no differences in cell survivalwere noted between BHK/hNGF and BHK/CONTROL cells.

These data indicate implantation of polymer encapsulated BHK cells,genetically-modified to secrete hNGF, prior to intrastriatal injectionsof QA, results in attenuation of the associated neurotoxicity. QA aloneproduced a marked striatal atrophy together with loss of ChAT andNADPH-d-positive neurons. Implantation of BHK/hNGF cells decreased theoverall extent of the lesion produced by QA. The attenuation ofQA-induced toxicity was associated with a preservation of ChAT andNADPH-d-positive neurons within the striatum. Associated with thehistological protection produced by hNGF was a significant attenuationof apomorphine-induced rotational behavior.

EXAMPLE 23 Treatment of Neural Degeneration in Non-Human Primates UsingEncapsulated Genetically Modified BHK Cells

In this example we grafted BHK cells into the lateral ventricle offornix-transected nonhuman primates and assessed the ability of polymerencapsulated NGF-secreting cells to prevent the degeneration of primatecholinergic basal forebrain neurons which normally occurs followingaxotomy.

BHK-hNGF Cell Line Production.

The BHK-hNGF cell line was produced as described in Example 22.

Encapsulation Procedure

BHK-hNGF cells were encapsulated as described in Example 22.

Surgical Procedures

Young adult cynomolgus (Macaca fascicularis) monkeys of both sexes (N=8;4-6 kg) were used in this study. Animals were tranquilized with ketamineHCL (10 mg/kg, im) and intravenous lines were secured for fluidadministration. The animals were intubated via the orotracheal methodand anesthesia maintained throughout the procedure with isoflurane(1.5-2.0%). Animals were placed on a heating pad to maintain bodytemperature and electrocardiogram leads placed to monitor heart rate andrhythm. To facilitate relaxation of the brain and minimize trauma due toretraction pressure, mannitol (0.250 mg/kg, iv) was administeredimmediately prior to the craniotomy.

Unilateral transections of the left fornix were performed using an openmicrosurgical approach developed by Kordower and Fiandaca (1990). Aftersecuring the animals in a Kopf stereotoxic headframe, a midline incisionwas made in the scalp and the skin retracted laterally. The medialattachment of the temporalis muscle was mobilized and a surgical drillused to create a parasagittal bone flap (size=1.5 cm×4.0 cm) whichexposed the frontal superior sagittal sinus. The dura was retracted anda self-retaining retractor used to expose the interhemispheric fissure.The parasagittal bridging veins were coagulated where needed tofacilitate retraction of the cerebral hemisphere. With the aid of asurgical microscope, arachnoid adhesions were divided. When necessary,veins overlying the corpus callosum were coagulated. The corpus callosumwas longitudinally incised exposing medial subcortical structures fromthe septum and head of the caudate rostrally through the foramen ofMonro caudally. At the level of the foramen of Monro, the fornix iseasily visualized as a discrete 2-3 mm wide white fiber bundle. Thefornix was initially transected using a ball dissector then the cut endsof the fornix were suctioned to ensure completeness of the lesion.

Following the transection of the fornix, individual BHK cell-containingcapsules were manually placed within the lateral ventricle with fineforceps between the head of the caudate and the septal nucleus. A totalof 5 capsules were implanted in each animal oriented in a row in therostrocaudal direction. The capsules abutted the caudate and septum,remained upright, and did not require to be secured further. Fouranimals received BHK/hNGF capsules, three received BHK controlcell-loaded capsules and one monkey received a fornix transection but notransplant. With hemostasis achieved, the dura was reapproximated, thebone flap was sutured back in place and the galea and skin was suturedusing routine methods. All animals received antibiotics (Cefotaxime, 50mg/kg, IM) for 4 days postoperatively.

Histology

Twenty-three to twenty-eight days following surgery, animals wereanesthetized as described above. Two-three ml of CSF was obtained fromeither the lumbar region (N=1 BHK control; 2 BHK/hNGF) or cistema magna(N=2 BHK control; 2 BHK/hNGF) to assay hNGF levels. Animals were thenplaced into the stereotoxic frame, the previously prepared bone flap wasremoved, the cerebral hemisphere retracted and the BHK cell-loadedcapsules removed. Immediately following removal of the capsules, animalswere transcardially perfumed using a peristaltic pump with 1 liter ofphosphate-buffered saline (pH=7.4) containing 1 ml of Heparin followedfixation with 3.5 lifers of 4% paraformaldehyde. The brains were blockedin the transverse plane following fixation, stored in 25% bufferedsucrose (pH 7.4) and refrigerated for 5-7 days.

Frozen sections were cut (30μm) on a sliding knife microtome and everyseventh section through the septal/diagonal band complex was processedimmunocytochemically for choline acetyltransferase (CHAT) and the lowaffinity p75 NGF receptor (NGFr). Immunocytochemical labeling wasconducted according to previously published protocols and brieflyconsisted of: 1) overnight incubation in PBS containing 0.4% Triton+2%normal serum, 2) 48 hour incubation in the primary CHAT polyclonalantibody (Chemicon; 1:10,000), or NGFR monoclonal antibody (generouslyprovided by Dr. Mark Bothwell; 1:20,000), 3) overnight rinse in PBS+0.2%Triton, 4) 6×5 minute rinse in PBS followed by a 1.5 hour incubation inthe appropriate biotinylated secondary IgG antibody (Vector; 1:100), 5)6×5 minute rinses in PBS+Triton, 6) incubation with “Elite”Avidin-Biotin complex (Vector, 1:1000) for 1.5 hours, 7) 3×10 minutesrinses in PBS, 8) incubation in the chromagen solution containing 0.05%3,3′ diaminobenzidine, 2.5% nickel ammonium sulfate dissolved in 0.1%Tris buffer for 5 minutes followed by hydrogen peroxide (0.01%) for 5minutes. The reaction was terminated by 3×1 minute rinses in PBS.Sections were mounted, dehydrated in alcohols and coverslipped. Controlsections were processed in an identical manner except the primaryantibody solvent or an irrelevant IgG was substituted for the primaryantibody. Adjacent sections were stained with hematoxylin and eosin (H &E) to aid in cytoarchitectonic delineation.

To verify the completeness of the lesion, sections through thehippocampus were processed histochemically for the visualization ofacetylcholinesterase (AChE) using the procedure of Hedreen and coworkers( ). Sections were incubated for 1 hour in a solution (pH 5.0)containing 100 mM sodium acetate (65 ml), 50 mg acetythiocholine iodide,100 mm sodium citrate (5 ml) 30 mm copper sulfate (10 ml), 15 ml dH20, 5mM potassium ferricyanide (4 ml) and 0.001Mtetraisopropylpyrophophoramide (iso-OMPA). After 3×10 minute washes insodium acetate buffer, the sections were incubated for 1 minute in 4%ammonium sulfide. After 5×10 minute washes in sodium nitrite, thesection were incubated for 1 minute in a 0.05% silver nitrate solution.After 5×10 minute washes in sodium nitrate, sections were mounted,dehydrated, and coverslipped as before. For control, sections wereprocessed in an identical manner except that acetylthiocholine iodidewas omitted from the incubation medium.

For quantification of cholinergic cell loss, the number of ChAT andNGFr-positive neurons were manually counted within the medial septum(MS) and vertical limb of the diagonal band (VLDB) at a totalmagnification of 10×. ChAT-positive neurons on the midline were excludedfrom this analysis. Representative sections (4 per brain) locatedapproximately 200 μM from each animal were used for this analysis. Forstatistical analysis, the numbers of neurons ipsilateral to the lesionwere expressed as percentages of neurons contralateral to the lesion.Student's t test was used to determine differences between theBHK-control and BHK-hNGF groups.

NGF-Induced Neurite Outgrowth

Conditioned media (CM) from encapsulated BHK-control and BHK-hNGF cellswas passed thru a 0.2 μm filter and added to PC12A cells grown onstandard tissue culture 6 or 24 well plates at a concentration of200,000 cells/ml to test for the presence of hNGF in the CM. All mediumconditioning and neurite outgrowth assays were performed in 5% CO2 andat 37° C. As a positive control, 2.5S mouse NGF was added to some of thewells to induce neuritic extensions (50 ng/ml). The PC12A cells werescored for neurite processes that were ≧3 times the length of the cellbody diameter after a period of 1-4 days. In addition, the rate ofneurite induction, and the stability of the neurites was observed and acomparison was made between the culture conditions.

NGF ELISA

Quantification of hNGF released from both encapsulated BHK-hNGF cellswas performed as described in Example 22.

Results

The BHK cell-loaded devices were retrieved from the lateral ventricles23-28 days following implantation with little to no host tissue adheringto the capsules. The level of hNGF produced by the capsules prior toimplantation was 21.4±2.0 ng/capsule/24 hr and 8.3±1.2 ng/capsule/24 hrin the retrieved capsules. The BHK-control capsules produced nodetectable hNGF.

The BHK-hNGF cell-loaded devices were left in situ in 1 of theBHK-control animals for fixation to demonstrate placement of the devicesand observe the host tissue response. All capsules were placed withinthe lateral ventricle and abutted both the head of the caudate and thelateral septum. The host response to these capsules was excellent, withlittle evidence of immune cells surrounding the capsules. Aproliferation of small to moderate sized blood vessels and a mildgliotic response was observed around the capsules particularly at theinterface between adjacent capsules.

In retrieved capsules containing BHK-\hNGF cells, few adhering hostcells were found on the capsule wall and a large number of viable BHKcells, evenly distributed at high density, were present within thepolymeric device. Numerous mitotic figures were observed throughout allof the cell-loaded capsules. Morphologic analysis of H & E-stainedacrylate sections revealed that encapsulated cell survival wasequivalent between the control and BHK-hNGF cell-loaded capsules.

In all animals, histological examination revealed that the left fornixwas completely transected while the contralateral fornix remainedintact. The completeness of the lesion was verified by demonstratingthat within the hippocampus ipsilateral to the lesion there was aprofound reduction in AChE staining. Some remaining AChE-positive fiberswere observed diffusely distributed within the lesioned hippocampus andlittle reduction of staining was observed within the inner third of themolecular layer of the dentate gyrus.

No differences in the extent of the fornix lesion or the loss ofcholinergic neurons were observed between the animal that received notransplant and those receiving control BHK cells. Accordingly, the datafrom these groups were combined. In these animals, a significantreduction was observed in the number of cholinergic neurons ipsilateralto the lesion. NGFr-positive neurons were decreased 54% within the MSand 30% within the VLDB compared to the intact side. The loss ofChAT-positive neurons paralleled the loss of NGFr labeled neurons andwas 53% within the MS and 21% within the VLDB. While many survivingcholinergic neurons ipsilateral to the lesion appeared normal, othersappeared shrunken, pale and dystrophic. In contrast, BHK-NGF transplantsresulted in a significant attenuation of the loss of cholinergic neuronsfollowing fornix transection. Analysis of NGFr-positive neurons revealeda modest loss neurons within the MS (19%) and VLDB (7%).

Similarly, ChAT-immunoreactive neurons in NGF-treated animals weredecreased only 20% in the MS and 7% in the VLDB. Cholinergic neurons inthe NGF-treated animals were generally larger and appeared to be moreintensely labeled than those in the BHK-control animals. Sectionsthrough the septum of the NGF-treated animals revealed a dense sproutingof cholinergic fibers within the septum in both the ChAT and NGFrpreparations. These fibers ramified against the ependyinal lining of theventricle adjacent to the transplant site and were particularlyprominent within the dorsolateral quadrant of the septum correspondingto the normal course of the fornix. This sprouting of cholinergic fiberswas not observed in animals receiving BHK-control implants. Despite theprevention of the loss of cholinergic neurons and induction of sproutingof these same neurons, hNGF was not detectable (limit of detectionequals 25 pg) within CSF taken from either lumbar and cistema magnataps.

These findings support use of polymer-encapsulated cell therapy intreating neurodegenerative diseases such as Alzheimer's disease wherebasal forebrain degeneration is a consistent pathological feature.

EXAMPLE 24 Treatment of Neural Degeneration in Aged Non-Human PrimatesUsing Encapsulated Genetically Modified BHK Cells

Many mammalian species, including humans, are known to undergo neuronalloss as a natural consequence of the aging process. Aged non-humanprimates were used in this experiment to evaluate whether aged neuronswould respond to growth factors in a manner similar to neurons inyounger animals. Fimbria fornix lesions were performed in aged non-humanprimates according to the method described in example 23. Theencapsulated cells, surgical procedure and analytical methods were thesame as reported in example 23. The animals used in these studies were24-29 year old Rhesus monkeys. Similar protection of cholinergic basalforebrain neurons to that observed in example 23 was also obtained inthese older animals.

EXAMPLE 25 Delivery of a Putative Parkinson's Factor (GDNF) into the RatCNS Using Encapsulated BHK Cells

Parkinson's disease is a progressive neurodegererative disorder ofunknown etiology in which midbraid dopaminergic neurons are graduallylost, leading to movement disorders and eventually death. A growthfactor, glial cell line-derived neurotrophic factor, (GDNF) has beendescribed that exhibits an apparent trophic activity for midbraindopaminergic neurons in vitro (Lin et al., Science, 260, p. 1130(1993)). These experiments evaluated the in vivo effect on dopaminergicfunction of delivery of rGDNF using encapsulated genetically modifiedBHK cells.

PCR Cloning of GDNF

Reverse transcription-PCR was performed on total RNA extracted from E18rat brain. The PCR primers that were used were synthesized based on thepublished sequence (Lin et al., Science, 260, pp. 1130 (1993)) forcloning into the pNUT expression vector (Baetge et al. Proc. Natl. Acad.Sci., 83, pp. 5454-58 (1986)).

The cDNA was subcloned into the pNUT vector and restriction digests weredone to determine insert orientation. A sense and antisense clone wereselected and then prepared for transfection. A modified calciumphosphate transfection method was used to introduce the expressionvectors into BHK cells. The cells were then selected in methotrexate for6-8 weeks to amplify the vector and the gene expression.

Cell Culture

The BHK cells were cultured in standard cell cultured medium containingfetal bovine serum. Conditioned medium was obtained by adding a defined,serum-free medium to both the sense and antisense BHK cell lines for 48hours. The primary mesencephalic tissues were dissected from E15 ratfetuses and enzymatically dissociated and plated in 24 well plates(Nunc) in a serum-free, defined medium (Hycor) and incubated at 37° C.and 5% CO2. To assess the potential of the GDNF to enhance tyrosinehydrosine hydroxylase (TH) neuron survival, various amounts of theconditioned medium from both cell lines was added in separate wells tothe mesencephalic cultures for up to 3 weeks.

Dopaminergic neuron survival was assayed by staining the cultures for THafter treatment for 1, 2 or 3 weeks. Immunocytochemistry was performedusing a mouse monoclonal antibody for TH (IncStar) followed by detectionwith a Vector Mouse ELITE kit and visualized using diaminobenzidine.Cell counts of TH-positive neurons was done using an inverted microscopewith bright field optics.

In general, the TH+ neurons in the cultures treated with the rGDNFexhibit an increased arborization of processes, increased THimmunoreactivity and in general a more robust appearance.

GDNF mRNA and protein expression was verified in the BHK-rGDNF(sense)cell line using Northern blot analysis and with a primary ventralmesencephalic neuronal bioassay for dopaminergic neuron survival(TH-positive).

To determine whether rGDNF has any effect on dopaminergic function invivo, both cell lines (sense or antisense) or untransfected BHK cellswere encapsulated in immunoisolatory polymeric devices and implantedunilaterally into the striatum of normal Lewis rats. In those animalsreceiving rGDNF, behavioral alterations including movement asymmetrieswere detected after a 1-2 mg/kg dose of amphetamine. No such asymmetrywas seen in the control animals.

A repeated measures analysis of variance was conducted including celltype, and with amphetamine dose and turning direction included asrepeated measures. Rats were more active with increasing doses ofamphetamine, the main effect of amphetamine dose was statisticallysignificant, F(2,32)=36.90, p=0.0001. The main effect of movementdirection was also statistically significant, F(1,6)=19.81, p=0.0004.This asymmetry in movement direction increased as the drug doseincreased, the drug dose by turning direction interaction wasstatistically significant, F(2,32)=8.43, p=0.001, and the movementasymmetry was significantly larger in the rats receiving encapsulatedGDNF-transfected bHK cells than in the rats receiving the encapsulatednon-transfected bHK cells, the movement direction by cell typeinteraction was statistically significant, F(1,16)=24.74, p=0.0001. Itshould be noted that the direction of the movement asymmetry in the ratsimplanted with encapsulated GDNF-transfected BHK cells was such thatthey moved more in the direction contralateral to the implant than inthe direction ipsilateral to the implant.

1. An implantable immunoisolatory vehicle for providing a biologicallyactive product or function to an individual, the immunoisolatory vehiclecomprising: (a) a core comprising a volume in excess of 1 μl and atleast about 10⁴ living cells dispersed in a biocompatible matrix formedof a hydrogel, said cells being capable of secreting a selectedbiologically active product or of providing a selected biologicalfunction to an individual; and (b) an external diffusional surfacejacket surrounding said core formed of a biocompatible hydrogel materialfree of said cells projecting externally thereof, wherein the hydrogelmaterial does not ionically bond to a polymer of opposite charge on thecore during formation of the jacket and wherein the jacket and the coreform an interface free of an intermediate linking layer said jackethaving a molecular weight cutoff below the molecular weight ofsubstances essential for immunological rejection of the cells butpermitting passage of substances between the individual and core throughsaid jacket required to provide said biological product or function.